SURVEY AND SUMMARY: Recent advances in the elucidation of the mechanisms of action of ribozymes

Yasuomi Takagi; Masaki Warashina; Wojciech J. Stec; Koichi Yoshinari; Kazunari Taira

doi:10.1093/nar/29.9.1815

SURVEY AND SUMMARY: Recent advances in the elucidation of the mechanisms of action of ribozymes

Takagi, Yasuomi;Warashina, Masaki;Stec, Wojciech J.;Yoshinari, Koichi;Taira, Kazunari 2001-05-01 00:00:00 © 2001 Oxford University Press Nucleic Acids Research, 2001, Vol. 29, No. 9 1815–1834 SURVEY AND SUMMARY Recent advances in the elucidation of the mechanisms of action of ribozymes 1 1 2 1 Yasuomi Takagi , Masaki Warashina , Wojciech J. Stec , Koichi Yoshinari and 1,3, Kazunari Taira * Gene Discovery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Science City 305-8562, Japan, Polish Academy of Science, Center of Molecular and Macromolecular Studies, Department of Bioorganic Chemistry, Sienkiewicza 112, 90-363 Lodz, Poland and Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Hongo, Tokyo 113-8656, Japan Received as resubmission February 15, 2001; Revised and Accepted February 27, 2001 ABSTRACT INTRODUCTION The cleavage of RNA can be accelerated by a number Naturally existing catalytic RNAs include hammerhead, hairpin, hepatitis delta virus (HDV) and Varkud Satellite (VS) of factors. These factors include an acidic group ribozymes; group I and II introns; and the RNA subunit of (Lewis acid) or a basic group that aids in the deprotona- RNase P (1–6). The structures of these catalytic RNAs are tion of the attacking nucleophile, in effect enhancing shown in Figure 1. In addition, recent structural and chemical the nucleophilicity of the nucleophile; an acidic analyses strongly suggest that the ribosomal RNA is a group that can neutralize and stabilize the leaving ribozyme (7–10) and the possibility that the RNA component group; and any environment that can stabilize the of the spliceosome might also be a ribozyme (11). pentavalent species that is either a transition state or Extensive efforts over the 15 years that followed the discovery of ribozymes (1,2) have revealed details of the a short-lived intermediate. The catalytic properties of mechanisms of the ribozyme-mediated cleavage (or ligation) ribozymes are due to factors that are derived from of RNA. Ribozymes have been considered to be ‘fossil mole- the complicated and specific structure of the cules’ that originated in a hypothetical prebiotic RNA world ribozyme–substrate complex. It was postulated and it is likely that elucidation of their mechanisms of action initially that nature had adopted a rather narrowly will enhance our understanding of the life processes of primi- defined mechanism for the cleavage of RNA. tive organisms (12–33). Since the earliest research on However, recent findings have clearly demonstrated ribozymes, it was assumed that all ribozymes are metalloen- the diversity of the mechanisms of ribozyme-catalyzed zymes that require divalent metal ions for catalysis and that all must operate by a basically similar mechanism. However, reactions. Such mechanisms include the metal- recent advances have revealed examples of cleavage by hairpin independent cleavage that occurs in reactions ribozymes that are independent of divalent metal ions (34–39). catalyzed by hairpin ribozymes and the general Thus, the various types of ribozyme appear to exploit different double-metal-ion mechanism of catalysis in reactions cleavage mechanisms, which depend upon the architecture of catalyzed by the Tetrahymena group I ribozyme. the individual ribozyme. Furthermore, it was proposed recently Furthermore, the architecture of the complex between that nucleobases in the HDV ribozyme might be candidates for the substrate and the hepatitis delta virus ribozyme participants in acid/base catalysis (40–42). allows perturbation of the pK of ring nitrogens of cyto- In addition, even hammerhead ribozymes, generally charac- terized as typical metalloenzymes, can no longer be unambig- sine and adenine. The resultant perturbed ring nitro- uously categorized (43,44). Recent findings indicate that the gens appear to be directly involved in acid/base hammerhead ribozyme might operate via a variety of cleavage catalysis. Moreover, while high concentrations of mechanisms, depending on the conditions of the reaction. monovalent metal ions or polyamines can facilitate Nevertheless, there is no doubt that RNA catalysts with groups cleavage by hammerhead ribozymes, divalent metal that are poorly functional under physiological conditions do ions are the most effective acid/base catalysts under cooperate with metal ions to exert their catalytic activity and physiological conditions. that many ribozymes can exploit divalent metal ions as cofactors *To whom correspondence should be addressed at: Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Hongo, Tokyo 113-8656, Japan. Tel: +81 35841 8828; Fax: +81 298 61 3019; Email: [email protected] The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors 1816 Nucleic Acids Research, 2001, Vol. 29, No. 9 Figure 1. The two-dimensional structures of various ribozymes. The ribozyme or intron portion is printed in green. The substrate or exon portion is printed in black. Arrows indicate sites of cleavage by ribozymes. (A) Left, the two-dimensional structure of a hammerhead ribozyme and its substrate. Outlined letters are conserved bases that are involved in catalysis. Right, the γ-shaped structure of the hammerhead ribozyme–substrate complex. (B–F) The two-dimensional struc- tures of a hairpin ribozyme, the genomic HDV ribozyme, a group I ribozyme from Tetrahymena, a group II ribozyme from Saccharomyces cerevisiae (aiγ 5) and the ribozyme of RNase P from Escherichia coli, respectively. and as stabilizers of their respective higher-order structures. considered to be roughly equivalent to the non-enzymatic The widespread potential utility of RNA molecules as catalysts hydrolysis of RNA, with inversion of the configuration at a and the events during reactions catalyzed by ribozymes, in phosphorus atom suggesting a direct in-line attack with devel- particular the actions of catalytic functional groups such as opment of a pentacoordinate transition state or intermediate. metal ions and pK -perturbed nucleobases, have generated The chemical cleavage requires two events, which can occur considerable interest (1–49). In this article we shall review either via a two-step mechanism or via a concerted mechanism various naturally existing ribozymes that cleave RNAs, (4,5,25,45). focusing mainly on the various mechanisms of catalysis. In the first step of the non-enzymatic hydrolysis of RNA (25,50–52), the 2′-OH attacks the adjacent scissile phosphate, acting as an internal nucleophile (transition state 1; TS1) (Fig. 2). CLEAVAGE OF THE PHOSPHODIESTER BOND In the second step, the 5′-oxygen of the leaving nucleotide is For the cleavage of RNA phosphodiester linkages, three types released to produce a 3′-end 2′,3′-cyclic phosphate and a of large ribozyme, namely, group I and II introns and the catalytic 5′-OH terminus (transition state 2; TS2). Of the two putative RNA subunit of RNase P, accept external nucleophiles (the 2′- transition states, TS2 is the overall rate-limiting state OH group of an internal adenosine in the case of the group II [i.e., attack by the 2′-OH on the phosphorus atom is easier than intron). By contrast, small ribozymes, such as hammerheads, cleavage of the P-O(5′) bond and, thus, TS2 always has higher hairpins, HDV and the VS ribozyme, use an internal nucleophile, energy than TS1] (25). This conclusion was confirmed in namely, the 2′-oxygen of the ribose moiety at the cleavage site, experiments with an RNA analog with a 5′-mercapto leaving with resultant formation of a 3′-terminal 2′,3′-cyclic phos- group. If the formation of the intermediate were the rate- phate. In general, ribozymes catalyze the endonucleolytic limiting step (i.e., if TS1 were a higher-energy state than TS2) transesterification of the phosphodiester bond, requiring struc- in the natural RNA, the phosphorothiolate RNA (RNA with a tural and/or catalytic divalent metal ions under physiological 5′-bridging phosphorothiolate at the scissile linkage) should be conditions. The reactions catalyzed by small ribozymes are hydrolyzed at a rate similar to the rate of the hydrolysis of the Nucleic Acids Research, 2001, Vol. 29, No. 9 1817 Figure 3. Possible catalytic functions of metal ions in the cleavage of a phos- phodiester bond. Metal ions can act as (a) a general acid catalyst, (b) a general base catalyst, (c) a Lewis acid that stabilizes the leaving group, (d)aLewis acid that enhances the deprotonation of the attacking nucleophile and (e)an electrophilic catalyst that increases the electrophilicity of the phosphorus atom. natively, hydrogen bonding between a metal-bound water molecule and the non-bridging oxygen might stabilize the Figure 2. The two-step reaction scheme for the hydrolysis of a phosphodiester charged trigonal-bipyramidal intermediate (or transition state). bond in RNA. First, the 2′-oxygen attacks the phosphorus atom, acting as an internal nucleophile, to generate the pentacoordinated intermediate or transi- Metal ions can function in several different ways as cofactors in tion state, TS1. The 5′-oxygen then departs from the intermediate to complete ribozyme-catalyzed reactions, as described above, and cleavage at TS2. TS1 can be stabilized by a general base catalyst and TS2 can proposed mechanisms for the reactions catalyzed by several be stabilized by a general acid catalyst, as illustrated at the summits of the energy diagram. These transition states can also be stabilized by the direct ribozymes have taken advantage of such functions. Moreover, binding of Lewis acids to the 2′-attacking oxygen and the 5′-leaving oxygen. it is difficult to imagine that a specific ribozyme might exploit multiple mechanisms (for example, coexistence in a reaction of the left structure and the central structure in Figure 3 at the natural RNA because the 5′-bridging phosphorothiolate transition state of a ribozyme-catalyzed reaction) under a linkage would not be expected to enhance the attack by the single set of physiological conditions. Significant aspects of 2′-oxygen (53). By contrast, if the decomposition of the inter- these functions of metal ions might be subsumed by nucleo- mediate were the rate-limiting step (i.e., if TS2 were a higher- bases if their pK values could be adjusted appropriately. The energy state than TS1) in the natural RNA, the phosphoro- full details of the mechanisms of action of metalloenzymes thiolate RNA would be expected to be hydrolyzed much more remain to be elucidated. rapidly than the natural RNA because the pK of a thiol is >5 units lower than that of the corresponding alcohol. Several groups have confirmed that the phosphorothiolate RNA is LARGERIBOZYMES significantly more reactive than the corresponding natural The group I intron, the group II intron and the RNA subunit of RNA in non-enzymatic hydrolytic reactions (25,45,54–56) RNase P are categorized as large ribozymes. Group I and II and, thus, TS2 is, indeed, always a higher-energy state than introns are found in bacteria and in the organelles of higher TS1. plants, fungi and algae (57,58). These introns are spliced out of their primary transcripts by a two-step mechanism [Fig. 4A(i) and POSSIBLE CATALYTIC FUNCTIONS OF METAL B(i)]. In the first step of splicing, the 5′ splice site is attacked IONS IN THE CLEAVAGE OF RNA by the 3′-OH of the external guanosine (group I). Alter- natively, it is attacked by the 2′-OH of the internal adenosine If ribozymes operate as metalloenzymes (4,5,15–25,27,45), the residue or by a hydroxide ion, in the case of hydrolysis (group possible catalytic functions of metal ions can be summarized II).Inthe second step,the 3′-OH of the 3′-end of the upstream as follows (Fig. 3). exon attacks the 3′ splice site to produce splicing products. · A metal-coordinated hydroxide ion might act as a general RNase P is an endonuclease that generates the mature base, abstracting the proton from the 2′-OH (Fig. 3b) or, alter- 5′-ends of tRNAs. In bacterial RNase P, the RNA subunit natively, a metal ion might act as a Lewis acid to accelerate the (RNase P ribozyme) has catalytic activity and the protein deprotonation of 2′-OH by coordinating directly with the 2′- component is thought to act only to facilitate the binding of the oxygen (Fig. 3d). anionic RNase P ribozyme to its substrate. However, mutations · The developing negative charge on the 5′-oxygen leaving in either the gene for the RNA or the gene for the protein can group might be stabilized by a proton that is provided by a inactivate RNase P in vivo, demonstrating that both compo- solvent water molecule or by a metal-bound water molecule as a general acid catalyst (Fig. 3a) or, alternatively, by direct nents are necessary for natural enzymatic activity. In the cleavage by the RNase P ribozyme, a scissile-site phosphate is coordination of a metal ion that acts as a Lewis acid catalyst attacked by a hydroxide ion to leave a 3′-oxygen and to (Fig. 3c). produce a 5′-phosphate terminus. · Direct coordination of a metal ion to the non-bridging oxygen might render the phosphorus center more susceptible All of these ribozyme reactions proceed with inversion of to nucleophilic attack (electrophilic catalysis; Fig. 3e) or, alter- configuration at a phosphorus atom (59–62), suggesting direct 1818 Nucleic Acids Research, 2001, Vol. 29, No. 9 Figure 4. A schematic representation of splicing reactions and the structures of transition states at each step. (A) The group I intron splicing reaction. (i) In the first step, the 3′-OH of the exogenous conserved G attacks the phosphorus at the 5′ splice site and generates the G-attached intron 3′–exon 2 intermediate and a free 5′ exon 1. In the second step, the 3′-OH of the 5′ exon 1 attacks the phosphorus at the 3′ splice site to produce ligated exons and the excised G-attached intron. (ii) 2+ The proposed chemical mechanism of the first step. The 3′-OH of the exogenous G is a nucleophile and the 3′-OH of the U is a leaving group. One of the Mg –1 ions [site (b)] coordinates with the 3′-OH of the G to activate the attacking group. The second [site (c)] coordinates with the 2′-OH of the G. The third [site (d)] coordinates with the pro-Sp oxygen to stabilize the transition state or the intermediate. The fourth [site (a)] coordinates with the 3′-OH of the U to stabilize the –1 leaving group. The 2′-OH also protonates the 3′-leaving oxygen of the U . It is not known whether or not the metal ion at site (d) is the same as those at the other –1 sites, (a), (b) and (c) (72). IGS represents the internal guide sequence. (B) The group II intron splicing reaction. (i) In the first step, the 2′-OH of an A residue that is conserved in the intron attacks the phosphorus at the 5′ splice site and generates an intron 3′–exon 2intermediateand afree5′ exon 1. In the second step, the free 3′-OH of the 5′ exon attacks the phosphorus at the 3′ splice site to produce ligated exons and an excised intron. SER indicates the spliced-exon reopening reaction. (ii) The proposed chemical mechanisms of the first and the second steps. In the first step, the 2′-OH of an intron A residue is the nucleophile and the 3′-OH 2+ of the 5′ splice site terminus is the leaving group. One Mg ion coordinates with the 3′-OH to stabilize the leaving group. Other coordinations and/or interactions remain to be clarified. In the second step, the 3′-OH of the C, the 5′ splice site terminus, becomes the nucleophile and the 3′-OH of the U is the leaving group. One 2+ Mg ion coordinates directly with the 2′-OH and the 3′-OH of the U. Other coordinations and/or interactions remain to be clarified. 2+ in-line attack with development of a pentacoordinate transition rule, a ‘hard acid’, such as a Mg ion, prefers to bind to a ‘hard state or intermediate (59–72). base’ oxygen atom rather than to a ‘soft base’ sulfur atom. By 2+ 2+ contrast, a ‘soft acid’, such as Cd or Zn ions, prefers to bind 2+ to a ‘soft base’ sulfur atom. A Mn ionis alsosofterthana THE MECHANISM OF REACTIONS CATALYZED BY 2+ Mg ion and, thus, the former can bind to a soft sulfur atom (as THE GROUP IINTRONRIBOZYME 2+ well as to a hard oxygen atom). This ability of Mn ions is believed to be the origin of the manganese rescue effect. In studies of the reactions mediated by the ribozyme from the Tetrahymena group I intron, detailed kinetic and thermo- Analysis of both the thio effect and of soft acid rescue 2+ 2+ 2+ dynamic analysis, combined with modifications at the atomic effects, such as the rescue effects of Cd ,Mn and Zn ions, level, helped to define the reaction mechanism of this has contributed significantly to our understanding of the catalytic ribozyme at the atomic level (18,63,66,68–72). Modification at mechanism of the first step of the reaction catalyzed by the the atomic level has generally involved replacement by a sulfur group I intron. Such analysis has revealed the importance of atom of an oxygen atom that has the potential to interact with a three to four independent metal ions, as shown in Figure 4A(ii) catalytically important metal ion. The observed reduction in (69,71). It is generally accepted that the group I intron is a 2+ the cleavage rate in the presence of Mg ions after such metalloenzyme that operates via the general double-metal-ion 2+ modification (the ‘thio effect’) and the observed restoration of mechanism of catalysis, in which a Mg ion at site (b) [see 2+ a normal cleavage rate in the presence of Mn ions (the Fig. 4A(ii) for locations of (a), (b), (c) and (d)] enhances the ‘manganese rescue effect’) have been taken as evidence that deprotonation of the 3′-OH of the guanosine nucleophile and a 2+ supports the direct coordination of the atom in question with a Mg ion at site (a) stabilizes the leaving 3′-bridging oxygen of metal ion. This phenomenon can be explained by the HSAB U in the transition state. In this case, the divalent metal ions –1 (Hardand Soft, Acidand Base)rule(73,74).Accordingtothis function as Lewis acids for activation of the nucleophile and Nucleic Acids Research, 2001, Vol. 29, No. 9 1819 stabilization of the leaving group by coordinating directly with result suggests that a metal ion at site (c) coordinates directly them (63). This mechanism corresponds, in the reactions cata- with the 2′-OH. lyzed by small ribozymes, to the central mechanism shown in In addition to the coordination of metal ions discussed Figure 3 with the stabilization of both TS1 and TS2 (Fig. 2) by above, other interesting interactions have been proposed. two metal ions. Linear free-energy analysis of the cleavage of oligonucleotide These details of coordination at the catalytic site were substrates with a series of 2′-substituents at U indicated that –1 derived from the following observations. The substitution of theeffect on therateof the 2′-OH group is larger than might be the 3′-oxygen of the guanosine nucleophile with a sulfur atom expected from simple inductive effects (81). The weaker reduced the rate of the reverse reaction in the presence of the electron-withdrawing 2′-OH enhanced the chemical cleavage 2+ hard acid, namely, Mg ions, and an efficient cleavage was step to a greater extent than did the more strongly electron- 2+ 2+ restored by Mn ions (66). This result suggests that a Mg ion withdrawing 2′-F atom of the corresponding 2′-deoxy-2′-fluoro at site (b) coordinates with the 3′-oxygen of the guanosine derivative. Therefore, the possibility was recently examined of a nucleophile to activate the first step. Next, the 3′-bridging symmetrical transition state, in which the 2′-OH of U might –1 phosphorothiolate substrate (3′-S substrate), in which the or might not interact with a metal ion [as observed at site (c) in 3′ leaving oxygen had been replaced by a sulfur atom, had a Fig. 4A(ii)] (71). Despite the absence of lone-pair electrons at dramatically reduced cleavage rate for the forward reaction in the 2′-NH group that need to interact with a metal ion, the 2+ thepresenceofMg ions. An efficient cleavage was restored + higher reactivity of the substrate with a 2′-deoxy-2′-NH 2+ 2+ by Mn ions (63,75). This result suggests that a Mg ion at group than that of the substrate with a 2′-OH group at U –1 site (a) in Figure 4A(ii) coordinates with the 3′-leaving oxygen suggested that interaction of a metal ion with the 2′-OH of U –1 during cleavage. These observations can be explained by the might not be important for catalysis by the group I intron 2+ double-metal-ion model, in which one Mg ion coordinates + ribozyme. The higher reactivity of the 2′-NH derivative with the nucleophile to activate the attacking group and the suggests that donation of a hydrogen bond from the 2′-group to 2+ other Mg ion coordinates with the 3′-leavingoxygentostabi- the neighboring 3′-leaving oxygen might allow specific stabiliza- lize the developing negative charge during RNA cleavage. tion of the transition state relative to the ground state, thereby 2+ The possibility of coordination of a Mg ion at site (d) in facilitating the chemical cleavage step. Figure 4A(ii) with the pro-Sp oxygen was suggested on the The 2′-OH of U ,the 2′-OH of A and the exocyclic amino –1 207 basis of the following experimental data. The RpS substrate, in group of G have been referred to as a catalytic triad (70). which the pro-Rp oxygen at the scissile phosphate had been However, the observation that the chemical cleavage step with replaced by sulfur, was cleaved at a modestly reduced rate a2′-NH derivative is faster than that with the substrate with a (76). By contrast, the SpS substrate, in which the pro-Sp 2′-OH (the natural substrate), despite the absence of lone-pair oxygen at the scissile phosphate had been replaced by sulfur, electrons at the 2′-NH group that can accept a hydrogen bond 2+ 3 had a drastically reduced cleavage rate in the presence of Mg from A -OH, suggests another possibility for the arrange- ions (71,75,77). Furthermore, the SpS/3′-S substrate, in which ment of active-site groups within this network of interactions not only the pro-Spoxygen but alsothe 3′-leaving oxygen had (71,72). been replaced by sulfur atoms at the same scissile phosphate, Even though the ribozyme-mediated chemical cleavage step was cleaved at a lower rate than the 3′-S substrate in the with the 2′-OH group at U (the natural substrate) is signifi- 2+ 2+ –1 presence of Mn ions on a background of Mg ions. An cantly (1000-fold) faster than that with 2′-H, with the metal- efficient cleavage of the SpS/3′-S substrate, with the double- 2+ 2+ 2+ binding site (a) in Figure 4A(ii) being occupied by a Mn ion, thio substitution, was restored by Zn or Cd ions, which are 2+ 2+ the rate constants for reactions with the 3′-S substrates are more thiophilic than Mn ions, on a background of Mg ions similar, irrespective of whether there is a 2′-OH or 2′-H at U . –1 (75). Thus, a thio effect seemed apparent at the pro-Sp oxygen, 2+ 2+ 2+ Moreover, in the presence of Mg ions, with the metal-binding and rescue both by Cd andbyZn ions was also evident. 2+ site (a) being unoccupied by a metal ion, the rate constants for These results suggested that a Mg ion(s) might coordinate reactions with the 3′-S substrates are similar with a 2′-OH or with the pro-Sp oxygen, as well as with the 3′-leaving oxygen. with a 2′-H at U , indicating that the 2′-OH at U does not –1 –1 However, in consideration of the above results, we should contribute significantly to the chemical cleavage of the note that an efficient cleavage of the SpS substrate, with phosphorus–sulfur bond with the 3′-mercapto leaving group. the single-thio substitution, could not be restored either by 2+ 2+ 2+ Since sulfur is a weaker acceptor of a hydrogen bond than is Mn ions or by Zn or Cd ions (75). This observation 2+ oxygen and, furthermore, since sulfur is a significantly better prevents us from ruling out the possibility that a Mg ion does leaving group than oxygen, the 3′-mercapto leaving group not coordinate with the pro-Sp oxygen in a direct manner suppresses the catalytic advantage provided by a hydrogen during the first step in cleavage by the group I intron ribozyme bond from the 2′-OH in the native transition state (71). (72). Further investigations are needed to determine whether 2+ The second step of the splicing reaction is catalyzed within direct coordination of a Mg ion occurs at site (d). the same catalytic site as the first step (18,82,83). Moreover, in Additional coordination has also been proposed at the 2+ 2+ thepresenceofMg ions, both the reverse reaction of the first catalytic site. A Mg ion at site (c) in Figure 4A(ii) might step and the forward reaction of the second step were inhibited interact directly with the 2′-OH of the guanosine, as suggested with the RpS substrate (note that the pro-Rpoxygenat these by experiments with a 2′-amino-2′-deoxyguanosine substrate and various metal ions in the ribozyme reaction (78–80). The steps corresponds to the pro-Sp oxygen in the forward reaction cleavage rate was reduced by replacement of the 2′-OH with of the first step). These observations indicate that the stereo- 2+ 2′-NH on a background of Mg ions. An efficient cleavage chemical requirements are the same in both reactions 2+ 2+ was restored by addition of soft Mn or Zn ions (78,79). This (60,63,76,84). Therefore, the mechanism of the second step is 1820 Nucleic Acids Research, 2001, Vol. 29, No. 9 2+ considered to be analogous to the mechanism of the first step cleaved faster in the presence of Mn ions than in the presence 2+ (18). of Mg ions at higher pH (at higher pH, the 2′-amino group It is clear that the analysis of thio effects, rescue experiments exists in a neutral form, as -NH , and not in the protonated + 2+ form, -NH )sinceaMn ion binds to the 2′-NH group better and other experiments with derivatives have contributed 3 2 2+ than a Mg ion (nitrogen is a softer base than oxygen and the significantly to our understanding of the mechanism of action 2+ 2+ of the large group I intron ribozyme of Tetrahymena.All the Mn ion is a softer acid than the Mg ion). This interaction with the 2′-oxygen at the second step involves a single metal available data appear to support the refined double-metal-ion ion in the transition state, as indicated by the fact that the mechanism of catalysis (18) that is shown in Figure 4A(ii). 2+ dependence on the concentration of Mn ions with the 2′- THE GROUP II INTRON RIBOZYME 2+ amino substrate on a background of Mg ions is consistent with a single-metal-ion exchange (85). In the splicing reactions catalyzed by the group II intron ribozyme (Fig. 4B), the first step was blocked when the RpS The 3′-S substrate reduced the cleavage rate of the second 2+ substrate was used (61) and a small thio effect was observed step by ∼100-fold in the presence of Mg ions, and the reac- 2+ 2+ with the SpS substrate (64), suggesting that the pro-Rpoxygen tion was rescued completely by the addition of Mn ,Co or 2+ makes an essential interaction in the transition state and that Cd ions in the tripartite assay (67,85). However, this the pro-Sp oxygen is also involved in some kind of interaction. substrate had no effect on the bimolecular cis-splicing assay in No cleavage of the 3′-S substrate was observed in the presence which the rate-limiting step appeared to be the conformational 2+ 2+ 2+ 2+ of Mg ions but the reaction was rescued by Mn ,Zn or rearrangement (67). This result indicates that Mg ions also 2+ 2+ Cd ions (67). This result indicates that a Mg ion acted to acted in the second step to stabilize the 3′-leavingoxygenby stabilize the 3′-leaving oxygen by direct coordination. direct coordination, as was the case in the first step. This inter- 2+ The second step was monitored by a tripartite assay (trans- action with the 3′-oxygen also involved a single Mn ion in the splicing), in which an oligonucleotide that corresponded to the transition state, as observed for the cleavage of the 2′-amino 3′-splice site was added after the formation of the ribozyme/ substrate (85). We should emphasize, however, that the prefer- 5′-exon RNA complex, because the second step might be ences of the 3′-S substrate for metal ions differed between the 2+ 2+ 2+ 2+ 2+ masked by the rate-limiting conformational rearrangement first step (Mn ,Zn or Cd ) and the second step (Mn ,Co 2+ between the first step and the second step that was observed in or Cd ), indicating the differences between the environments the bimolecular assay (cis-splicing). In contrast to the results of of metal ions at the two different transition states for each analysis of the first step, the cleavage of the RpS substrate was splicing step. strongly inhibited but the SpS substrate had essentially no The moiety that activates the attacking nucleophile in the inhibitory effect (85). The actual stereospecificity for the thio two independent splice steps of the reaction of the group II substitution is reversed between the first step and the second intron ribozyme remains to be identified but it is apparent that 2+ step (both steps were inhibited in the reaction with the RpS aMg ion binds to the leaving 3′-oxygen to stabilize the tran- substrate; note that the pro-Rpoxygeninthe forwardreaction sition state at each step. of the first step corresponds to the pro-Sp oxygen in the reverse reaction of the first step and in the forward reaction of the THE RNASE P RIBOZYME second step). Thus, the second step is not the reversal of the first step, unlike results for the group I intron ribozyme. Even In reactions catalyzed by the RNA subunit of bacterial RNase 2+ though the pro-Rp oxygen atom appears to make an essential P, there is a requirement for both divalent cations (e.g., Mg or 2+ + + interaction in the transition state, the nature of this interaction Mn ) and monovalent cations (e.g., K or NH ) (62). Mono- has not been defined since no rescue by thiophilic metal ions valent cations appear to be involved in the stabilization of the can be observed. structure during the cleavage reaction in the absence of The group II intron ribozyme can also hydrolyze the bond proteins in vitro. By contrast, divalent metal cations are between spliced exons [the spliced-exon reopening (SER) required for the chemical cleavage itself, and not only for reaction, which corresponds to the reverse reaction of the structural stabilization. During the chemical cleavage, a second step]. This SER reaction proceeds with the RpS hydroxide ion, activated by metal ions, is thought to act as a substrate but not with the SpS substrate (64). Since this step nucleophile (86,87). Though the details of the reaction mecha- would be expected to be blocked with the RpS substrate if it nism are not fully understood, it has been proposed that three 2+ followed the same reaction pathway as that of the first step of Mg ions participate in the transition state, because the slope the splicing reaction, the observed SER reaction supports the of the Hill plot for the cleavage rate versus the concentration 2+ 2+ conclusion that the second step is not the reversal of the first of Mg ions was 3.2, and that one of the catalytic Mg ions step, as mentioned above. coordinates directly to the pro-Sp oxygen at the scissile phosphate. The substitution of the 2′-OH of the leaving ribonucleoside 2′-Deoxy substitution at the cleavage site reduced the apparent 2+ (the U residue of the intron in Fig. 4B) at the 3′-splice site with number of bound Mg ions and decreased the apparent affinity 2+ a hydrogen atom reduced the rate of the second step ∼700-fold for Mg ions, suggesting that the 2′-oxygen might be one of 2+ (85). Moreover, even though substitution with a methoxy the Mg -binding sites. Furthermore, 2′-methoxy substitution group or a fluorine atom, respectively, reduced the rate simi- at the cleavage site decreased the cleavage rate, suggesting that larly to or significantly more than substitution with a hydrogen the 2′-OH might be involved in stabilizing the 3′-leaving atom, substitution with an amino group resulted in a rate that oxygen as the donor of a hydrogen bond (87). was ∼10-fold higher than that with a hydrogen atom (85). According to a recent report, the cleavage rate of the RpS 2+ These results suggest that the ability to donate a hydrogen bond substrate in the presence of Mg ions was at least 1000-fold from the 2′-OH group is important. The 2′-amino substrate was lower than the cleavage rate of the natural substrate. The Nucleic Acids Research, 2001, Vol. 29, No. 9 1821 reduction was, however, rescued by thiophilic metal ions, such certain pH between the ∆ pK values of metal ions in water and 2+ 2+ 2+ as Cd and Mn ions (background Mg ions are needed for the difference in the observed rates of cleavage in the presence rescue in the case of RNase P ribozyme from Bacillus subtilis), of the corresponding metal ions suggested a single-metal-ion 2+ suggesting the direct coordination of a metal ion to the pro-Rp mechanism in which Mg -hydroxide acts as a general base 2+ oxygen (88,89). Since the Hill coefficient for Cd rescue was catalyst (17). However, it was also noted that a general double- 1.8, it was proposed that two metal ions coordinate to the metal-ion mechanism, in which metal ions act as Lewis acids pro-Rp oxygen in a modified model, which is consistent with that coordinate directly to the 2′-OH and the 5′-leaving the two-metal-ion model of Steitz and Steitz (18). By contrast, oxygen, for activation of a nucleophile and for stabilization of the cleavage reaction was also blocked with the SpS substrate a developing negative charge on the leaving group, respec- (with reduction of the binding affinity of the substrate for the tively, might also explain reactions catalyzed by hammerhead ribozyme in the ground state in the case of the RNase ribozymes (20–22,24,25). Pribozyme from Escherichia coli) and the cleavage site was It should also be noted that, under extreme conditions (in the + + shifted in the 5’ direction. The reduced rate of cleavage of the presence of 1–4 M monovalent cations, such as Li ,Na and 2+ 2+ SpS substrate was not enhanced by Cd and Mn ions, an NH ), hammerhead ribozymes do not require divalent metal observation that suggests the possibility of a crucial role for the ions for catalysis (43). On the basis of this observation, some pro-Sp oxygen in stabilization of the transition state or that researchers have claimed that hammerhead ribozymes are not might be attributable to the steric exclusion of catalytic metal metalloenzymes (see below). ions (88,89). Similar effects of RpS and SpS substrates were HDV ribozymes are derived from the genomic and the anti- also observed with the eukaryotic nuclear RNase P ribozyme, genomic RNAs of hepatitis delta virus (105–108). In studies of in which the RNA is thought to be the catalytic component and reactions catalyzed by HDV ribozymes, three groups demon- to be evolutionarily related to the bacterial RNase P ribozyme strated recently that an intramolecular functional group, (90). However, no thio effect was observed in the case of namely N3 at C in the antigenomic HDV ribozyme and N3 at RNase P from plant chloroplasts, whose catalytic component C in the genomic HDV ribozyme, can, in fact, act as a true appears to be a protein (91). catalyst (40–42). However, with respect to the roles of these The 3′-S substrate also prevented cleavage at the correct site N3s, two different mechanisms, namely general base catalysis by the bacterial RNase P ribozyme and the cleavage site was and general acid catalysis, were proposed. In the former moved to the next unmodified phosphodiester bond in the scenario, it was proposed that the deprotonated N3 of C 5′-direction completely. The reduction in the cleavage rate was might be involved in cleavage as a general base that abstracts a 2+ 2+ not rescued by thiophilic Cd or Mn ions (92). While the proton from the 2′-OH to promote its nucleophilic attack on the absence of rescue by thiophilic metal ions does not reveal the scissile phosphate in the transition state of reactions catalyzed molecular nature of the inhibitory effects (the thio effect and by the antigenomic HDV ribozyme (41). In the latter case, it 2+ shifting of the cleavage site), prevention of binding of a Mg was proposed that the protonated N3 of C in the genomic ion to the 3′-leaving atom as a result of the thio substitution HDV ribozyme might act as a general acid to stabilize the provides one possible explanation. In addition, it is possible developing negative charge at the 5′-leaving oxygen and that a that several chemical and structural changes occur upon the metal ion might act as a general base (42). However, although introduction of a bulky sulfur atom. further investigations are required, there remains the possi- bility that the catalytic mechanism of the antigenomic ribozyme is the same as that of the genomic ribozyme (109). SMALL RIBOZYMES In discussion of the reaction catalyzed by the genomic HDV Hammerhead, HDV, hairpin and VS ribozymes are categorized as ribozyme, the importance has been emphasized of the neutraliza- small ribozymes because they are smaller than the ribozymes tion of the substantial negative charge that develops on the discussed above. Each of these naturally existing ribozymes 5′-leaving oxygen and the essential role of general acid catalyzes the endonucleotic cleavage of RNA via a mechanism catalysis in the cleavage of RNA (42). Such issues should be that involves nucleophilic attack by a 2′-OH group on the phos- relevant not only in the case of reactions catalyzed by HDV phorus of the neighboring phosphodiester bond, generating 5′-OH ribozymes but also in the case of reactions catalyzed by other and 2′,3′-cyclic phosphate termini (for reviews, see 4,25,93). small ribozymes since cleavage of the bond between phos- The cleavage reactions catalyzed by these ribozymes appear to phorus and the 5′-oxygen is the overall rate-limiting step, as proceed with inversion of the configuration at the phosphorus shown in Figure 2 (see below; 22,25,56). The efficient atom suggesting a direct in-line attack with development of a cleavage of a phosphodiester bond requires both the activation pentacoordinate transition state or intermediate (42,94–97). of the 2′-attacking oxygen and the stabilization of the The smallest of the naturally occurring catalytic RNAs that 5′-leaving oxygen. have been identified to date are the hammerhead ribozymes Hairpin ribozymes were originally derived from the minus (Fig. 1A), which were found in several plant viral satellite strand of the satellite RNA of tobacco ringspot virus (sTRSV), RNAs, a viroid RNA and the transcript of a nuclear satellite chicory yellow mottle virus type 1 (sCYMV1) and arabis DNA of a newt (98; for reviews, see 3,4). These ribozymes mosaic virus (sArMV) (110–113). Hairpin and hammerhead have been extensively investigated, in particular with respect ribozymes can also catalyze the ligation of cleaved products, to the mechanism of action of catalytic metal ions with the ligation efficiency being much higher for the hairpin (17,25,27,45,46). ribozyme than the hammerhead. The ligation reaction is Hammerhead ribozymes have a basic requirement for divalent thought to be the reverse of the cleavage reaction since it uses 2+ metal ions, such as Mg ions (5,6,17–22,24,25,27,45,99–104). the same termini as those produced upon cleavage. Hairpin In studies of the hammerhead reaction, the relationship at a ribozymes favor the ligation reaction rather than cleavage 1822 Nucleic Acids Research, 2001, Vol. 29, No. 9 (ligation occurs 10-fold faster than cleavage). By contrast, chemical cross-linking studies (Fig. 1A, right) (26,139–142). hammerhead ribozymes favor the cleavage reaction [cleavage Such studies indicate the involvement of two reversed-Hoogs- occurs ≥100-fold faster than ligation (47,114–116)]. The ratio teen G·A base pairs between G ·A and A ·G , aswellasa non- 8 13 9 12 of equilibrium constants (k /k ) can be explained by Watson–Crick A ·U base pair that is formed by a single cleavage ligation 14 7 the differences between entropies: the loss of entropy that hydrogen bond. These base pairs are followed by stem II and are occurs with ligation is smaller for the hairpin than for the stacked ‘coaxially’ onto the non-Watson–Crick A ·U base 15.1 16.1 hammerhead ribozyme, indicating that the more rigid hairpin pair, with resultant formation of a pseudo-A-form helix by structure undergoes a smaller change in dynamics on ligation stems II and III. Four sequential nucleotides (C U G A )form 3 4 5 6 than the more flexible hammerhead (117). Catalysis by hairpin a ‘uridine-turn’ motif, allowing the phosphate backbone to turn ribozymes in the absence of metal ions has been reported by and connect with stem I. The uridine-turn forms a catalytic several groups independently (34–39). Hairpin ribozymes can pocket into which the nucleobase at the cleavage site, namely be considered to be a distinct class of ribozymes that do not C , is inserted (133). The crystal structure of the enzyme– require metal ions as cofactors (118). The catalyst(s) seems to product complex of the hammerhead ribozyme has been deter- be a nucleobase(s). mined (137). The structure suggests that the distance between C and G /A in the transition state is smaller than previously The VS ribozyme originated from the mitochondria of 17 5 6 proposed and that dramatic conformational changes, which certain isolates of Neurospora (119). The reaction catalyzed by 2+ include C , occur in the pathway from the ground state to the the VS ribozyme requires a divalent cation such as the Mg transition state. ion (120). Some regions that are important for catalysis and some interactions between the phosphate backbone and metal Structural metal ions in reactions catalyzed by ions have been identified (121–123). The reaction appears to hammerhead ribozymes be independent of pH but the possibility exists for a conforma- tional change prior to cleavage that might mask a dependence It is generally accepted that the tertiary structures of RNA on pH (120,124). The catalytic group(s) in the cleavage molecules are stabilized by metal ions. The roles of metal ions reaction has not yet been unambiguously identified. in ribozyme-catalyzed reactions are of two distinct types: metal ions can act as catalysts during the chemical cleavage step, as shown in Figure 3; and they can also stabilize the REACTIONS CATALYZED BY HAMMERHEAD conformation of the ribozyme–substrate complex. The ion- RIBOZYMES dependent changes in the conformation of a hammerhead ribozyme can be easily followed by monitoring the influence Hammerhead ribozymes are among the smallest catalytic of metal ions on its electrophoretic mobility (26). The effects RNAs. The sequence motif, with three duplex stems and a of metal ions on the formation, upon subsequent addition of conserved core of two non-helical segments that are respon- 2+ Mg ions, of an active complex between a hammerhead sible for the self-cleavage reaction (cis-action), was first recog- ribozyme and its substrate can also be monitored by NMR nized in the satellite RNAs of certain viruses (3). Engineered spectroscopy (143–152). trans-acting hammerhead ribozymes, consisting of antisense sections (stem I and stem III) and a catalytic core with a Binding sites for metal ions have been identified by flanking stem–loop II section (Fig. 1A, left), have been used in capturing metal ions within the crystal structure, and such mechanistic studies and tested as potential therapeutic agents capture provides an indication of the importance of the metal 2+ (13,46). Hammerhead ribozymes cleave their target RNAs at ions in catalysis (99,132–136). It was proposed that a Mg ion specific sites, generating a 2′,3′-cyclic phosphate and a 5′-OH binds to the pro-Rpoxygenofthe 5′-phosphate of A (P9 phos- terminus. The NUH rule, where N can be any nucleotide and H phate) with further hydrogen bonding associated with N7 of 2+ can be A, U or C, was originally proposed to define sites of G (153–155). Another site for a Mg ion was localized in 10.1 cleavage, with the most efficient cleavage occurring at GUC the vicinity of the cleavage site. It was proposed that, at this 2+ triplets (125–130). However, the NUH rule was reformulated cleavage site, a Mg ion binds directly to the pro-Rpoxygenof into the NHH rule since other triplets, such as GAC and GCC, the scissile phosphate. Although this possibility remains to be can also be cleaved by a hammerhead ribozyme (131). confirmed, the function of this second metal ion near the Over the past few years, several attempts have been made to scissile phosphate has been proposed to be activation of the determine the overall global structure of hammerhead attacking 2′-OH in the transition state. The site of yet another ribozymes (99,132–137). Although initial structural studies metal ion has also been proposed. Such an ion would act as a indicated that possible configurations of the scissile phosphate switch that induces the conformational changes required to did not allow for an in-line attack mechanism, recent crystallo- achieve the transition state; it would be located adjacent to G graphic studies of a ribozyme with a product or with a modifi- in the catalytic core. This last putative site was identified from 3+ cation (known as a kinetic bottleneck modification) adjacent to an analysis of the kinetics of a Tb inhibition experiment and the cleavage site succeeded in trapping an intermediate that more the elucidation of the crystallographic structure of the complex closely resembled the transition state (137; reviewed in 138). (136). The coordination of a metal ion at this site in solution However, even in this case, the intermediate cannot be consid- was also investigated by lanthanide luminescence spectros- ered as a real transition-state intermediate. In all crystals of copy (156). An additional metal ion binding site in the ribozymes examined to date, a γ-shaped configuration has been hammerhead ribozyme has also been identified by PNMR identified, with stem I forming an acute angle with stem II, and spectroscopy (149). In this case, the metal ion is associated stems II and III being stacked coaxially to form a pseudo-A- with the A phosphate in the catalytic core with an apparent K 13 d form helix, in agreement with results inferred from studies of of 250–570 µ M. However, the exact role of this metal ion fluorescence energy transfer and from electrophoretic and remains unclear. It seems likely that it might be involved in Nucleic Acids Research, 2001, Vol. 29, No. 9 1823 2+ 2+ structural folding since a structural change was detected at this no preference was detected for either Mg or Mn ions site upon the binding of the metal ion. (100,101). However, the 5′-S almost-DNA substrate basically The importance of the binding of a metal ion at the P9 phos- consisted of DNA and the ribozyme reaction with this substrate had an unusually limited dependence on pH. Thus, phate, not only in the ground state but also in the transition we might expect that observed rates of reaction might reflect state, was demonstrated by kinetic analysis with a modified hammerhead ribozyme with a phosphorothioate modification steps other than the chemical cleavage step. In the case of a 5′-S RNA substrate consisting only of RNA, the rate of at this site (157,158) and, in parallel, by analysis with a ribozyme-mediated cleavage of the 5′-S RNA substrate in the ribozyme with an abasic mutation at this site (159,160). The 2+ 2+ binding of a metal ion (Cd )to the Rpsulfurofthe P9 phos- presence of Mg ions was higher by almost two orders of magnitude than that of cleavage of the natural substrate (56). If phorothioate in the transition state was much stronger than the TS1 were a higher energy state than TS2 in the ribozyme reac- binding in the ground state, suggesting the existence of addi- tional ligands for the metal ion in the transition state. More- tion with the natural substrate, the cleavage rate for the 5′-S RNA substrate should be similar to that for the natural substrate over, our own studies indicate strongly that the binding of a because the 5′-bridging phosphorothioate linkage would not be metal ion to N7 of G is catalytically important but not indis- 10.1 pensable: cleavage still occurred with a minimally modified expected to enhance the attack by the 2′-OH (53). By contrast, if TS2 were a higher energy state than TS1 in the ribozyme ribozyme, in which N7 of G was merely replaced by C7 10.1 reaction with the natural substrate, we would expect that the (introduction of an N7-deazaguanine residue to prevent the metal ion from binding to this site) (161). Cleavage was rate of cleavage of the 5′-S RNA substrate would be much higher than that of the natural RNA substrate because a retarded, with ∼30-fold reduction in the rate of cleavage by the mercapto group is a better leaving group than a hydroxyl modified ribozyme. By contrast, a 1000-fold reduction in the cleavage rate resulted from the introduction of an Rp-phos- group. On the basis of these considerations, the results indicate that TS2 is a higher energy state than TS1 in the reactions of phorothioate at the P9 site (157). hammerhead ribozymes with natural substrates, as indicated in It was proposed very recently that the first metal ion that Figure 2. binds to the P9 phosphate in the ground state shifts toward a non-bridging pro-Rp oxygen at the scissile phosphate during Catalytic metal ions in reactions catalyzed by the reaction and binds to this pro-Rp oxygen in the transition hammerhead ribozymes: single-metal-ion mechanisms state. This scenario is consistent with the prediction that additional binding to this P9 metal ion must occur in the tran- In the case of the proteinaceous enzyme RNase A, which does sition state, as mentioned above. Furthermore, it was also not require metal ions as cofactors, the acid/base catalysts are proposed that the metal ion at P9 must be involved directly in provided by two histidine residues within the catalytic pocket the chemical cleavage step, acting as a base catalyst in the tran- (Fig. 5A). Such acid/base functionality can, in principle, be 2+ sition state (27), despite the fact that the P9 metal ion is located replaced by Mg -bound water molecules. The generally at a distance of ∼20 Å from the scissile phosphate in the ground accepted mechanism of hammerhead ribozyme reactions is a state. However, these proposals have been questioned by other single-metal-ion mechanism (27,45,99–103). In this proposed 2+ investigators (162,163). mechanism, the hydroxide ion of a hydrated Mg ion acts as a 2+ 2+ Although it was predicted that a hydrated Mg ion should general base to deprotonate the attacking 2′-OH; the Mg ion participate directly in catalysis, acting as a base catalyst in coordinates directly to the pro-Rp oxygen at the scissile phos- deprotonation of the 2′-OH of C , no crystal structure has been phate, acting as an electrophilic catalyst in TS1; and it is not a obtained with a trapped metal ion located at this cleavage metal ion but a proton that acts as a general acid to stabilize position that might confirm the direct involvement of such a TS2 (Fig. 5B). metal ion in deprotonation of the 2′-OH (137). The single-metal-ion mechanism is supported by the fact that no metal ion was found close to the 5′-leavingoxygeninthe The rate-limiting departure of the 5′-oxygen in reactions original crystallographic structure of a freeze-trapped confor- catalyzed by hammerhead ribozymes mational intermediate of a hammerhead ribozyme (99). Based In the non-enzymatic hydrolysis of a natural RNA, the upon the crystal structure and a molecular dynamic simulation, cleavage of the P-O(5′) bond is the overall rate-limiting step in a different single-metal-ion mechanism was proposed as hydrolysis. By contrast, attack by the 2′-OH on the phosphorus follows (102,103). It was suggested that the involvement of atom is the rate-limiting step with a 5′-S substrate that contains just one metal ion in the transition state might be sufficient for 2+ a phosphorothiolate substitution (54–56,164,165) since, as cleavage. In this model, one Mg ion coordinates simultane- mentioned above, the latter is hydrolyzed much more rapidly ously and directly to the pro-Rpoxygenand to the 2′-attacking than the natural substrate (Fig. 2). The same technique as that oxygen at the cleavage site, acting as a Lewis acid to enhance used to draw these conclusions was used to determine the rate- the deprotonation of the 2′-OH and the subsequent attack by limiting step in reactions catalyzed by hammerhead ribozymes. the nucleophile on the phosphorus atom and/or to stabilize the It was reported that, in the hammerhead-catalyzed cleavage transition state. In addition, it was also proposed that one of the reaction, the departure of the 5′-leaving group is not rate- outer-sphere water molecules that surrounds the metal ion limiting and that a metal cofactor does not interact with the might be located at a position such that it can act as a general leaving group (45,100,101). This conclusion was based on acid to donate a proton to the 5′-leaving group (102,103) experiments with the 5′-S almost-DNA substrate, that was an Another model for cleavage by hammerhead ribozymes was oligodeoxynucleotide substrate that contained a 5′-bridging proposed that was based on the results of molecular dynamic phosphorothiolate linkage adjacent to one ribonucleotide at the studies (166). This model involves two metal ions but it is cleavage site (45). No appreciable thio effect was observed and reminiscent of the single-metal-ion mechanism. In this model, 1824 Nucleic Acids Research, 2001, Vol. 29, No. 9 to the pro-Rp oxygen at the scissile phosphate bond (5,25,162,168,169). Studies of solvent isotope effects and kinetic analysis of a modified substrate (phosphorothiolate; 5′-S substrate), with a 5′-mercapto leaving group at the cleavage site, provided strong support for the double-metal-ion mechanism of catalysis (20,22,25). The overall transition state structure in the hammerhead cleavage reaction is TS2, regardless of whether the reaction proceeds via a concerted one-step mechanism or via a two-step mechanism with a stable pentacoordinated intermediate. Our analysis with a 5′-S substrate demonstrated that it is important that any enzyme that catalyzes the hydrolysis of RNA should stabilize TS2 by donating a proton to the 5′-leaving oxygen or by ensuring coordination of a metal ion to the leaving oxygen. This acid catalysis by a metal ion is also supported by a 2+ recently determined crystallographic structure, in which a Co ion was located close to the 5′-leaving-oxygen atom of the scissile phosphate (134). Figure 6A shows experimentally derived profiles of pH versus rate for reactions in H Oand D O (20,25,169). The 2 2 magnitude of the apparent isotope effect (ratio of rate constants in H Oand D O) is 4.4 and the profiles appear to support the 2 2 2+ possibility that a proton is transferred from (Mg -bound) water molecules. However, careful analysis led us to conclude that a metal ion binds directly to the 5′-oxygen. Since the concentration of the deprotonated 2′-oxygen in H Oshould be higher than that in D O at a fixed pH, we must take into account D2O H2O this difference in pK ,namely, ∆ pK (pK –pK ), when we a a a a analyze the solvent isotope effect of D O (20,25,169,170). We Figure 5. (A) The mechanism of cleavage by ribonuclease A. Two imidazole residues function as general acid–base catalysts. (B) The single-metal-ion can estimate the pK in D O from the pK in H Ousing the a 2 a 2 mechanism proposed for cleavage by the hammerhead ribozyme. One metal linear relationship shown in Figure 6B (6,25,169–172). If the ion binds directly to the pro-Rp oxygen and functions as a general base cata- 2+ pK for a Mg -bound water molecule in H O is 11.4, the ∆ pK a 2 a lyst. (C) The double-metal-ion mechanism proposed for cleavage by the ham- is calculated to be 0.65 (Fig. 6B, red line). Then, the pK in merhead ribozyme. Two metal ions bind directly to the 2′-and 5′-oxygens. a D O should be 12.0. Demonstrating the absence of an intrinsic isotope effect (k /k = 1), the resultant theoretical curves H2O D2O two metal ions are bridged by a hydroxide ion (the µ -hydroxo- closely fit the experimental data, with an apparently ∼4-fold 2+ bridged Mg cluster). One of the metal ions, located near the difference in observed rate constants (Fig. 6A). This result scissile phosphate, binds to the pro-Rp oxygen to act as an indicates that no proton transfer occurs in the hammerhead – 2+ electrophilic catalyst. The bridging OH between the two Mg ribozyme-catalyzed reaction in the transition state and supports 2+ ions abstracts the proton from the Mg -bound water molecule. the hypothesis that the metal ions function as Lewis acids. In 2+ Then the activated hydroxide ion associated with the Mg ion Figure 6A, the apparent plateau of rate constants above pH 8 deprotonates the proximal 2′-OH at the cleavage site to acti- reflects the disruption of the active hammerhead complex by vate the nucleophile, acting as a base catalyst. the deprotonation of uridine and guanosine residues. As described in the previous section, formation of TS2 is the The double-metal-ion mechanism is also supported by rate-limiting step in non-enzymatic reactions (25,54–56). results reported by Pontius et al. (21) and Lott et al. (24). They Thus, TS2 must somehow be stabilized energetically for effective 2+ pointed out the minimal likelihood of base catalysis by Mg - catalysis. Therefore, the hypothesis that reactions catalyzed by bound hydroxide that deprotonates the attacking 2′-OH and the hammerhead ribozymes involve only a general base is insuffi- strong likelihood of Lewis acid catalysis by the direct coordi- cient. 2+ nation of a Mg ion with the attacking 2′-OH, which enhances the deprotonation of the nucleophilic 2′-OH. Their suggestions Catalytic metal ions in reactions catalyzed by were based on the inverse correlation between the rate of hammerhead ribozymes: double-metal-ion mechanisms cleavage and the pK of the added metal ions. Their argument Double-metal-ion mechanisms, in which two metal ions are was as follows. One of the observations used to support the involved in the chemical cleavage step, have been proposed by single-metal-ion mechanism of catalysis (Fig. 5B) is that the numerous groups of investigators (5,18–22,24,25,104). From lower the pK , the higher is the cleavage rate at a given concen- molecular orbital calculations and kinetics analysis, our group tration of metal ions. Although metal ions with lower pK 2+ postulated that the direct coordination of Mg ions with the values might be present at higher concentrations in the form of attacking or the leaving oxygen might promote formation or solvated metal hydroxides at a given pH, such ions should be cleavage of the P-O bond, with these ions acting as Lewis acids correspondingly weaker bases and, therefore, they should be (Fig. 5C) (5,20,22,25,167). Moreover, we excluded the less able to remove the proton from the 2′-OH. As a result, the possible coordination of metal ions, as electrophilic catalysts, effect of concentration would be reduced by the effect of Nucleic Acids Research, 2001, Vol. 29, No. 9 1825 Figure 6. (A) The dependence on pH of the deuterium isotope effect in the hammerhead ribozyme-catalyzed reaction. Green circles show rate constants in H O; yellow circles show rate constants in D O. Solid curves are experimentally determined. The apparent plateau of cleavage rates above pH 8 is due to disruptive 2+ effects on the deprotonation of U and G residues. Dotted lines are theoretical lines calculated from pK values of hydrated Mg ions of 11.4 in H O and 12.0 in a 2 D O and on the assumption that here is no intrinsic isotope effect (α = k /k =1; where α is the coefficient of the intrinsic isotope effect). The following 2 H2O D2O (pKa(base) – pL) (pL – pKa(acid)) equation was used to plot the graph of pL versus log(rate): log k = log (k ) – log[1 + 10 ] – log[1 + 10 ]. In this equation, k is the rate obs max max constant in the case of all acid and base catalysts in active forms: in H O, k = k ; and in D O, k = k = k /α.(B) The isotope effects on the acidities 2 max H2O 2 max D2O H2O D2O H2O 2+ (pK – pK ) of phenols and alcohols as a function of their acid strengths (pK ). The pK of hydrated Mg ions in H O is 11.4, and the red arrow indicates a a a a 2 2+ the isotope effect of 0.65 that results in the pK of hydrated Mg ions in D O being 12.0. The pK of the N3 of cytosine in H O is 6.1 and the blue arrow indicates a 2 a 2 the isotope effect of 0.53 that results in the pK of N3 of cytosine in D O being 6.6. a 2 basicity. In other words, the dependence on pK cannot be ion, coordinates not only with the 5′-leaving oxygen but also, adequately explained by the hypothesis that the solvated metal directly, with the 2′-attacking oxygen [(c) in Fig. 7A]. 3+ hydroxide acts as a base in catalysis. By contrast, deprotona- Although the La ion, which is more positively charged than 2+ tion of the 2′-OH can be greatly accelerated by its direct the Mg ion, might enhance the deprotonation of 2′-OH and, binding to a metal ion, in particular, a metal ion with a rela- as a result, might increase the equilibrium concentration of 2′-O , tively low pK , because the pK of a 2′-OH with a bound metal it would also decrease the nucleophilicity of 2′-O toward the a a ion can be reduced by several units. Such arguments are further electropositive phosphorus. Because the coordination of a supported by ab initio molecular orbital calculations (173). trivalent metal ion at this position reduces the nucleophilicity of the resulting 2′-O much more dramatically than would be Further evidence for a double-metal-ion mechanism was expected for a series of divalent ions and since this negative based on an analysis of the effects of changes in the concentra- 3+ effect is greater than that of an induced higher concentration of tion of La ions in the presence of a fixed concentration of – 3+ 2+ 3+ 2′-O , a higher concentration of La ions decreases the overall Mg ions (24). Analysis of the effects of added La ions rate of cleavage [(c) in Fig. 7A]. yielded a bell-shaped curve, with activation and then inhibition of cleavage (Fig. 7A). Under the conditions of the experiment, However, it has been demonstrated that the binding of a 3+ 2+ La and Mg ions competed for coordination to restricted metal ion to the pro-Rp oxygen of the phosphate moiety of sites that are catalytically important for cleavage by the nucleotide A andtoN7 ofnucleotide G is critical for effi- 9 10.1 3+ 3+ ribozyme. At lower concentrations of La ,aLa ion rather cient catalysis, despite the considerable distance (∼20 Å) 2+ than a Mg ion coordinates to the 5′-leaving oxygen and between the P9 phosphate and the scissile phosphate in the absorbs the negative charge that accumulates at that position in ground state (27,99,128,132–134,157–160). In earlier discus- 3+ 3+ the transition state [(b) in Fig. 7A]. The fully hydrated La ion sions of the above-mentioned La -titration issue (24), it was 2+ 3+ has a pK of 9, which is >2 lower than that of Mg .Whenthe difficult to completely exclude the possibility that La ions 3+ leaving oxygen is directly coordinated with a trivalent La ion, might replace the metal ion at the P9 site and, as a result, might it is a better leaving group than when coordinated with a create the conditions represented by the bell-shaped curve 2+ divalent Mg ion. This difference results in a decline in the shown in Figure 7A. In order to clarify this situation, we examined relative energy of TS2 and acceleration of cleavage. However, a chemically synthesized hammerhead ribozyme (7-deaza-R34) 3+ 3+ 2+ at higher concentrations of La ,aLa ion, instead of a Mg that included a minimal modification, namely, an N7-deaza- 1826 Nucleic Acids Research, 2001, Vol. 29, No. 9 obviously, completely free from potential artifacts due to a 2+ Mg ion coordinated at G . Thus, our results strongly 10.1 support the proposal that a double-metal-ion mechanism is operative in the cleavage reaction that is catalyzed by hammer- head ribozymes (161). Catalytic metal ions as electrophilic catalysts The involvement of a metal ion in a strong interaction with the non-bridging pro-Rp oxygen of the scissile phosphate is generally accepted and has been supported by results from many groups (27,94–97,99,102,103,158,174–176), even though such involvement remains a matter of controversy in light of the work from our laboratory (5,6,25,104,162,168,177). It has been proposed that this metal ion acts as an electrophilic catalyst in the reaction catalyzed by hammerhead ribozymes (Fig. 3e) 2+ on the basis of results of a rescue experiment with Mn ions (94–97), with further support from molecular dynamic studies (102,103,174) and spectroscopic analysis (175). Replacement of the pro-Rp oxygen with sulfur at the cleavage site of a substrate molecule (to yield the RpS substrate for a hammerhead ribozyme) resulted in a large thio effect that 2+ 2+ was relieved by replacement of Mg ions with Mn ions, 2+ which have higher affinity than Mg ions for sulfur. This 2+ observation led to the general conclusion that a Mg ion is directly coordinated with the pro-Rp oxygen. In this arrange- ment, the bound metal ion can act as an electrophilic catalyst and, therefore, the proposed mechanism is very attractive as an explanation for the catalytic activity of metalloenzymes. However, as we have suggested previously, observations of the 3+ thio effect and manganese rescue do not, by themselves, prove Figure 7. Titration with Ln ions. The hammerhead ribozyme reaction was 2+ examined on a background of Mg ions. (A) Data obtained by Lott et al. (24). that the direct coordination of a metal ion with the pro-Rp The proposed binding of metal ions is illustrated. (B) Data obtained by Naka- oxygen at the cleavage site occurs in hammerhead ribozyme- matsu et al. (161). An unmodified ribozyme (R34; red curve) and a modified catalyzed reactions (5,25,168,177). Moreover, although results ribozyme (7-deaza-R34; blue curve) were used. The rate constants were nor- of a reinvestigation of the thio effect using another thiophilic 3+ malized by reference to the maximum rate constant ([La ]=3 µ M). Reactions 2+ metal ion, namely the Cd ion, were used to support the were performed under single-turnover conditions in the presence of 80 nM ribozyme and 40 nM substrate at 37°C. proposed direct coordination of the metal ion with the pro-Rp oxygen of the scissile phosphate (27,158,176), we did not find the argument totally convincing. guanine residue in place of G (Fig. 7B, blue curve) (161). 10.1 In studies that we designed as part of an attempt to explain We compared the kinetic properties of this ribozyme with the thio effect and cadmium rescue by mechanisms that do not those of the parental ribozyme (R34 in Fig. 7B, red curve). involve the direct coordination of a metal ion at the pro-Rp Kinetic analysis revealed that replacement of N7 by C7 at G 10.1 oxygen (162), we found that the rate of ribozyme-catalyzed reduced the catalytic activity but only to a limited extent cleavage of the RpS substrate was ∼1000-fold lower than that (∼30-fold). The most important result, however, was that 2+ of the natural substrate in the absence of thiophilic Cd ions 7-deaza-R34 also yielded a bell-shaped curve upon addition of 2+ and that the addition of the soft Cd ions to the reaction on a 3+ La ions to the reaction mixture. Moreover, the apparent K 2+ 2+ background of hard Mg or Ca ions restored the efficient for the replacement was the same for 7-deaza-R34 and for the cleavage of the RpS substrate (Fig. 8A). Furthermore, at high parental ribozyme R34 (Fig. 7B). These results indicated that 2+ 2+ concentrations of Cd ions on a background of Ca ions, the 2+ binding of a Mg iontoN7ofG was catalytically important 10.1 rate of cleavage of the RpS substrate was similar to that of the but not indispensable for hammerhead ribozyme-mediated natural substrate. 3+ catalysis and, furthermore, they confirmed that the La titra- Although such results might appear to prove the direct coor- tion method, as reported by von Hippel’s group, does indeed dination of a metal ion with the pro-Rp oxygen, we reached a allow monitoring of the actions of catalytic metal ions at the 2+ completely different conclusion. It is true that Cd ions at cleavage site that are directly involved in catalysis. While such 2+ higher concentrations replaced the background Ca ions, and results do not, by themselves, completely exclude the possi- 2+ that, above the apparent dissociation constant of the Cd ions bility that other hypothetical metal-binding sites might have an (K ), the RpS substrate was cleaved at a fixed rate because dapp 2+ 2+ allosteric effect on catalytic activity (136,156,174), the data the Ca ion(s) had been fully replaced by a catalytic Cd 2+ do, at least, increase the likelihood that catalysis by hammer- ion(s). However, it should be noted that Cd ions at higher 2+ head ribozymes involves a double-metal-ion mechanism since concentrations similarly replaced the background Ca ions in 2+ the involvement of a Mg ion at N7 of nucleotide G can be the reaction with the natural substrate and, moreover, that the 10.1 ignored. The data from our experiments with 7-deaza-R34 are, natural substrate (PO) was cleaved at a fixed rate that was identical Nucleic Acids Research, 2001, Vol. 29, No. 9 1827 known to interact with a sulfur atom with a significantly higher affinity (two orders of magnitude higher) than with a hard oxygen atom (162). Estimates can be made of the respective kinetic and thermodynamic parameters in the thermodynamic 2+ cycle (Fig. 8B), as follows. Since the pre-existing Ca ion(s) 2+ within the ribozyme–substrate complex was replaced by a Cd ion(s) with the identical K for both the natural and RpS dapp 2+ substrates, the affinity of the binding of the added Cd ion(s) to each respective complex in the ground state must be the same [K = K ]. Moreover, the cleavage rate in the d GS(PO) d GS(PS) 2+ presence of saturating Cd ions was the same for both the –1 natural and RpS substrates [k = k =1min ]and cleave(PO) cleave(PS) the rate of the non-enzymatic cleavage of the P-O bond in phosphate and phosphorothioate moieties is known to be similar [k = k ; see 178,179]. Because the thermodynamic (PO) (PS) box must be closed (Fig. 8B) and since three out of four param- eters, namely, K , k and k, are the same for both the dGS cleave natural and the RpS substrate, it is apparent that the remaining 2+ parameter (the affinity of the Cd ion for the transition state complex) must also be the same for both substrates [K = d TS(PO) K ], irrespective of whether the cleavage site includes a d TS(PS) regular phosphate or a modified Rp-phosphorothioate moiety. In the case of a modified ribozyme in which the pro-Rp oxygen at the P9 phosphate was replaced by sulfur (RpS-P9 2+ ribozyme), the affinity of Cd ions was higher for the RpS-P9 ribozyme than for the natural ribozyme (27,157,162) [K for dapp the RpS-P9 ribozyme (25 µ M) was smaller than K for the dapp 2+ natural ribozyme (220 µ M) in the presence of 10 mM Mg 2+ ions (157)]. Therefore, a Cd ion does indeed interact with the Rp sulfur at the P9 phosphate but, contrary to the conclusion 2+ reached by other investigators, the Cd ion does not interact with the sulfur atom at the Rp position of the scissile phos- phate, neither in the ground state [K = K ]nor in the 2+ Figure 8. (A) Titration with Cd ions. The hammerhead ribozyme reaction d GS(PO) d GS(PS) 2+ transition state [K = K ]. Thus, it is appropriate to was examined on a background of Ca ions. The substrate had a normal phos- d TS(PO) d TS(PS) phate (natural substrate; blue) or an Rp-phosphorothioate group (RpS sub- emphasize, yet again, that observations of the thio effect and strate; red) at the cleavage site. Solid curves indicate the rate constants for cadmium rescue by themselves are not sufficient to prove that 2+ Cd -associated reactions. Dotted curves indicate the observed rate constants. the direct coordination of a metal ion with the pro-Rpoxygen (B) Thermodynamic boxes for the cleavage of the natural substrate (blue) and at the cleavage site occurs in hammerhead ribozyme-catalyzed the RpS substrate (red) by the hammerhead ribozyme. R, ribozyme–substrate complex with all metal-binding sites occupied except the exchange site(s) reactions. examined here; M, the metal ion(s) that binds to the exchange site(s) examined Numerous experimental results have indicated that the here; K and K , the intrinsic dissociation constants for binding of metal dGS dTS substitution of only one sulfur atom for an oxygen atom has a ion(s) to the exchange site(s) examined here in the ground state and in the tran- major steric effect and that, in many cases, such substitution sition state, respectively; k , the rate of ribozyme-catalyzed cleavage with cleave 2+ Cd ion(s) at the exchange site(s) examined here; and k, the rate of non-enzy- changes the mode of reaction (64,72,88,89,92,149,179–184). matic cleavage. The observed rate constants can be described in terms of With a 3′-S or SpS substrate, the site of cleavage by the RNase pseudo equilibrium constant K s for the formation of the transition state (‡) P ribozyme shifted in the 5′-direction from the modified site from the ground state (k =[k T/h]K , in which k is Boltzmann’s constant, T B d B (the expected cleavage site) to the adjacent unmodified phos- is absolute temperature and h is Planck’s constant). phodiester linkage (88,92), as discussed above. With an SpS substrate, the cleavage site in the group II intron-catalyzed to the rate of cleavage of the RpS substrate (PS) above the K SER reaction also shifted in the 5′-direction (64). A hybridized dapp 2+ 2+ orbital of oxygen or sulfur consists of 2s,2p atomic orbitals or for Cd ions [k = k ]. Clearly, Cd ions enhanced cleave(PO) cleave(PS) 3s,3p atomic orbitals, respectively. The third periodic orbital is the rate of cleavage not only of the RpS substrate but also of the 2+ known to be much bigger than the second one. This difference natural substrate, an indication that Cd ions replaced bound in bulkiness might be responsible for the prevention of 2+ Ca ions even in the case of the natural substrate. Furthermore, the correct positioning of the scissile bond at the active 2+ the K for Cd ions during cleavage of the RpS substrate dapp site. Furthermore, in the case of hammerhead ribozymes, it is was the same as that during cleavage of the natural substrate possible that disruption of the symmetry of the non-bridging [K = K , where GS is the ground state]. d GS(PO) d GS(PS) oxygen by the thio substitution might itself affect the structure 2+ Why was the affinity for Cd ions the same for both the RpS and activity of the ribozyme (179). Introduction of a sulfur and the natural substrate? We would expect the RpS substrate to atom can also result in a significant change in the structure of 2+ have a higher affinity for Cd ions, if the metal ion were directly the complex between the hammerhead ribozyme and its 2+ coordinated with the pro-Rp oxygen, because the soft Cd ion is substrate (150). 1828 Nucleic Acids Research, 2001, Vol. 29, No. 9 2+ 2+ Rescue experiments with Mn or Cd ions do not provide general acid catalysis, as shown by the blue curve in Figure 9A evidence that unequivocally supports electrophilic catalysis in (without a metal hydroxide acting as a general base). This type 2+ hammerhead ribozyme-catalyzed reactions. Thus, Mg ions of profile clearly demonstrates that the observed pK of 6.1 for that catalyze the nucleophilic attack by the 2′-oxygen or that self-cleavage in the presence of divalent metal ions (Fig. 9B, stabilize the 5′-leaving oxygen have no kinetically detectable green curve) reflects the pK of a general acid rather than that 3+ function as simultaneous electrophilic catalysts in hammer- of a general base. It is noteworthy that [Co(NH ) ] inhibited 3 6 2+ head ribozyme-catalyzed reactions, as shown in Figure 5C. the Mg -catalyzed reaction in a competitive fashion, a result 3+ that suggests that [Co(NH ) ] might bind to the same site as 3 6 2+ the functional Mg ion with outer-sphere coordination (note HDV RIBOZYME-CATALYZED REACTIONS 3+ that [Co(NH ) ] does not ionize to yield base catalyst [B:]) 3 6 (42). The similar rate constants determined in the presence of Studies by three groups have revolutionized our understanding 2+ 2+ Ca ions and of Mg ions are also consistent with the action of the mechanism of HDV ribozyme-catalyzed reactions (40–42). of a hydrated metal ion as a Brönsted base rather than as a For the cleavage of phosphodiester bonds, the nucleophile Lewis acid in the reaction catalyzed by the HDV ribozyme must be deprotonated and the leaving group must be proto- (21,42). nated or stabilized by a functional group(s). As illustrated in Figure 4, the developing negative charges in the transition state There was a significant, apparent D O solvent isotope effect in the group I and group II intron-catalyzed reactions are stabi- over the entire range of pH values, confirming that transfer of lized by direct interactions with metal ions. A similar mecha- a proton occurs in the transition state [the ratio of the observed nism is possible for hammerhead ribozyme-catalyzed rate constants, k /k , being as high as 10 (42)]. obs(H2O) obs(D2O) reactions, as shown in Figure 5C. The novel finding with Moreover, since the observed pK for self-cleavage corre- respect to the mechanism of catalysis by the genomic HDV sponded to that for the general acid and since the overall rate- ribozyme is that the pK of cytosine 75 (C ) is perturbed to limiting step appeared to be the cleavage of the bond between a 75 neutrality in the ribozyme–substrate complex and, more the phosphorus and the 5′-leaving oxygen (Fig. 2), the trans- importantly, that C acts as a general acid catalyst in combina- ferred proton in the transition state must have been derived tion with a metal hydroxide which acts as a general base cata- from C . Under the conditions of the measurements, the pK 75 a lyst (Fig. 9). Discovery of this phenomenon provided the first of C in H O was 6.1 and, thus, the estimated ∆ pK was 0.53, 75 2 a direct demonstration that a nucleobase can act as an acid–base as indicated by the blue arrow in Figure 6B. Indeed, in the pL catalyst in an RNA. As a result, as shown by the green curve in profile (pL = pH or pD) in Figure 9B, the pK in D Oisshifted a 2 Figure 9B, the curve that represents the dependence on pH of upward by ∼0.4 ± 0.1 pH units, consistent with the estimated the self-cleavage of the precursor genomic HDV ribozyme has value (42). If we take the pK of C as 6.1inH Oand 6.5 in a 75 2 2+ a slope of unity at pH values below 7 (the activity increases D O and the respective pK values for Mg -bound water to be 2 a linearly as the pH increases, with a slope of +1). Then, at 11.4 and 12.0; and if we assume that the value of the intrinsic higher pH values, the observed rate constant becomes insensi- D O solvent isotope effect is 2 (k /k = 2), we can generate 2 H2O D2O tive to pH. theoretical curves for HDV-catalyzed reactions in H O (green curve) and D O (yellow curve), as shown in Figure 9B. The The slope of unity below pH 7 is consistent with an increase, good agreement of the theoretical curves with the experimen- with pH, in the relative level of the metal hydroxide [B:], tally determined curves (indicated by solid lines in Fig. 9B) which acts as the general base upon deprotonation, and a (42) supports a scenario wherein transfer of a proton does constant amount of the functional protonated form of C [AH], indeed occur from protonated C in the P-O(5′) bond-cleaving which acts as the general acid. The slope of zero from pH 7 to transition state (TS2; Fig. 9C). This conclusion is different pH 9 indicates that the relative level of the metal hydroxide from the conclusion in the case of hammerhead ribozymes [B:] increases (Fig. 9A, red curve), while the relative level of because, in the case of hammerhead ribozymes, the observed, protonated C [AH] decreases by the same amount (Fig. 9A, apparent isotope effect (Fig. 6A) can be fully explained by the blue curve) (42). In agreement with this interpretation, when difference in relative levels of the active species in H Oand in C was replaced by uracil, the resultant C75U mutant, which 75 2 D O without invoking any intrinsic D O solvent isotope effect was unable to assist in the transfer of a proton, did indeed lack 2 2 (k /k =1;Fig.6A). catalytic activity. However, the activity of the C75U mutant H2O D2O 2+ was restored by the addition of imidazole, whose protonated As described above, the HDV ribozyme requires a Mg ion form, the imidazolium ion, is known to act as a good proton for efficient catalysis. The possibility of the coordination of the 2+ donor (41,42). Another mutant, C75A, in which the ring Mg ion with the non-bridging oxygen during the ribozyme- nitrogen N1 at A has a slightly lower pK than the corre- mediated cleavage was examined with RpS and SpS substrates 75 a sponding ring nitrogen N3 of C , supported self-cleavage (185). Determination of the rates of the cleavage steps revealed activity, albeit with reduced efficiency. The observed pK of that the cleavage rates for both substrates were almost the same the C75A mutant was slightly lower than that of the wild-type as that for the natural substrate irrespective of the presence of 2+ 2+ C ribozyme, supporting the interpretation in Figure 9A. Mg ions or Mn ions. These results indicate the absence of a Further convincing evidence for this model comes from the thio effect and the possibility for manganese rescue in the observation that, in the absence of divalent metal ions (in the HDV ribozyme-catalyzed reaction. The association constants 2+ absence of base [B:], see Fig. 9A) and in the presence of a high for Mg ions and the ribozyme–substrate complex were also concentration of monovalent cations (1 M NaCl, 1 mM almost the same for the two substrates. This result supports the 2+ EDTA), the logarithm of the observed rate constant decreased hypothesis that no direct coordination of the Mg ion with any with pH with a slope of –1, as shown by the blue curve in non-bridging oxygen atoms occurs at the scissile phosphate Figure 9B. This observation is consistent with exclusively during cleavage in HDV ribozyme-catalyzed reactions (185), Nucleic Acids Research, 2001, Vol. 29, No. 9 1829 Figure 9. Reactions catalyzed by the genomic HDV ribozyme. (A) Fractions of the active species [AH] that acts as an acid catalyst (blue) and the active species [B:] that acts as a base catalyst (red), respectively. The pK of the acid catalyst is 6.1 and that of the base catalyst is 11.4 in H O. The theoretical curve for H O in (B) was produced a 2 2 by the multiplication of these two curves. (B) Dependence on pH of the deuterium isotope effect in the HDV ribozyme-catalyzed reaction. Green circles, rate constants in H O; yellow circles, rate constants in D O; solid curves, experimental data; dotted curves, theoretical data calculated using the equation in Figure 6 and pK values for C 2 2 a 75 2+ and for hydrated Mg ions of 6.1 and 11.4 in H O and 6.5 and 12.0 in D O, respectively, assuming α = 2. The blue curve is a pH profile in 1 M NaC1 and 1 mM EDTA in 2 2 the absence of divalent metal ions. (C) Energy diagram for cleavage of its substrate by an HDV ribozyme. The rate-limiting step in the reaction with the natural substrate is the cleavage of the P-(5′-O) bond. The structures of transition states TS1 and TS2 are also shown. P(V), the pentacoordinate intermediate/transition state. 1830 Nucleic Acids Research, 2001, Vol. 29, No. 9 2+ and that base catalysis involves the Mg ion with outer-sphere For efficient cleavage, the hairpin ribozyme seems to require not coordination (42). only activation of an attacking nucleophile but also stabilization of the leaving group since the cleavage mechanism is the All the available data support the reaction mechanism for ‘5′-leaving type’, as are the cleavage mechanisms of the HDV HDV ribozyme-catalyzed reactions that is shown in Figure 9C. and hammerhead ribozymes, which involve stabilization of In this model, the donation of a proton is favored and the model TS2. Hairpin ribozymes probably exploit a nucleobase(s) involvesanacidwithanoptimal pK of 7 under physiological instead of divalent metal ions for stabilization of TS2. conditions (42). By contrast, the self-cleavage of the HDV ribozyme in the absence of divalent cations, but in the presence of high concentrations of monovalent cations, suggests that REACTIONS CATALYZED BY RIBOZYMES THAT DO monovalent cations at high concentrations can replace divalent NOTINVOLVE DIVALENT METAL IONS cations in the tertiary folding of the RNA. Thus, divalent Many ribozymes are considered to be metalloenzymes, cations are not absolutely essential for the folding or cleavage 2+ requiring divalent metal ions for efficient catalysis. However, activity of the HDV ribozyme even though a functional Mg cleavage by hairpin ribozymes occurs in the absence of divalent ion does participate in cleavage under physiological condi- metal ions and some small ribozymes can catalyze cleavage in tions. These features appear to be unique among the mecha- the absence of divalent metal ions but in the presence of mono- nisms of action of known ribozymes. It should be emphasized + + + valent cations, such as Na ,Li and NH .Such ribozymes that, since the overall rate-limiting step is the cleavage of the have been called non-obligate metalloenzymes (43,44). It has P-O(5′) bond (TS2), the acid catalysis provided by protonated been suggested that a dense positive charge rather than divalent C is mandatory in reactions catalyzed by the HDV ribozyme metal ions is the general and fundamental requirement for (Fig. 9C). catalysis by a ribozyme (43). Nucleobases are new candidates for acid/base catalysts, as described above for the HDV THE HAIRPIN RIBOZYME ribozyme. In the case of hammerhead ribozymes, typically categorized as metalloenzymes, it seems that reactions can also 2+ The roles of Mg ions in catalysis have been discussed for be catalyzed by monovalent cations but the concentrations of naturally existing ribozymes, such as group I and II introns, such cations have to be very much higher than that of divalent RNase P, HDV and hammerhead ribozymes. The hairpin metal ions if cleavage is to occur at a similar rate. Monovalent ribozyme is exceptional in that the reaction catalyzed by the cations might stabilize the transition state, in particular the hairpin exploits a metal ion in only a passive role. Strong overall rate-limiting formation of TS2, in the same way as experimental evidence for this statement is provided by the divalent metal ions, by providing a dense positively charged observation that the reaction proceeds efficiently in the presence environment at high concentrations. If this is the case, mono- 3+ of [Co(NH ) ] , polyamines or aminoglycoside antibiotics, 3 6 valent cations would be very ineffective under physiological and in the absence of additional metal ions (34–39). The pK of conditions, at least in the case of reactions catalyzed by 3+ the coordinated NH groups of [Co(NH ) ] is estimated to be 3 3 6 hammerhead ribozymes. >14 (186). Moreover, it is an exchange-inert complex (the Important differences between monovalent cations and divalent –10 –1 ligand exchange rate is 10 s ) (186) and it can replace a fully metal ions include not only differences in positive-charge 2+ 3+ hydrated Mg ion since both [Co(NH ) ] and the fully 3 6 density but also differences in the geometry of hydration of the 2+ hydrated Mg ion have octahedral symmetry and similar radii. 2+ ions. Divalent metal ions, such as Mg ions, have octahedral 3+ Thus, it seems unlikely that [Co(NH ) ] functions as a + 3 6 coordination, while monovalent metal ions, such as Na and general base or a Lewis acid to deprotonate the 2′-OH for + Li ions, likely have tetrahedral coordination on the basis of the 3+ nucleophilic attack and it is likely that [Co(NH ) ] supports 3 6 valence bond theory (186,188). These features of monovalent the structure of the RNA appropriately in the same way as the metal ions might not be suitable geometrically for formation of 2+ hydrated Mg ions do. a catalytically competent structure in the transition state such 2+ Other evidence supporting the putative passive (non-cata- as the one shown in Figure 5C (with Mg ions being replaced + + lytic) role of metal ions is that ribozyme-catalyzed cleavage in by Na or Li ions). Therefore, it is likely that a very high 2+ thepresenceofMg ions exhibits no preference for the RpS concentration of such monovalent cations would be required to versus the SpS substrate (34). This result suggests that non- stabilize the overall transition state TS2. Thus, it is very difficult bridging oxygen atoms at the scissile phosphate do not interact to envision, for example, that a hammerhead ribozyme might with metal ions, at least in any direct manner, during cleavage. use monovalent cations as a catalytic cofactor under physio- Additional evidence for a passive role comes from the observa- logical conditions in vivo. Under physiological conditions, it is tion that the ribozyme reaction can occur in films of partially probable that hammerhead ribozymes are metalloenzymes that hydrated RNA in the absence of divalent cations (39). This require divalent cations for catalysis. result indicates that all elements essential for catalytic function are provided by the folded RNA itself. Taken together, all the CONCLUSIONS results support the hypothesis that a nucleobase(s) functions as a general acid/base catalyst. The profile of pH versus rate for The mechanisms exploited by naturally existing ribozymes hairpin ribozymes has two pK values (5.4 and 9.8), with a flat appear to be more diverse than originally anticipated. It is clear, pH-independent region at neutral pH (35). The importance of however, that at least two effective catalysts are necessary to the exocyclic 2-amino group of G in the substrate has been facilitate the overall pathway for the efficient hydrolysis of reported (187). Efforts are being made to identify a nucleo- RNA. In the case of the group I intron ribozyme, two metal base(s) that might act as a general base/acid catalyst. ions function as Lewis acids by coordinating directly with the Nucleic Acids Research, 2001, Vol. 29, No. 9 1831 8. Nissen,P., Hansen,J., Ban,N., Moor,P.B. and Steitz,T.A. (2000) The nucleophile and the leaving group. In the case of the group II structural basis of ribosome activity in peptide bond synthesis. Science, intron ribozyme, a metal ion functions as a Lewis acid to stabilize 289, 920–930. the departure of the 3′-oxygen and the attacking nucleophile is 9. Muth,G.W., Ortoleva-Donnelly,L. and Strobel,S.A. (2000) A single activated by certain interactions with the ribozyme. In the case adenosine with a neutral pK in the ribosomal peptidyl transferase center. Science, 289, 947–950. of the RNase P ribozyme, it has been proposed that two metal 10. Cech,T.R. (2000) Structural biology. The ribosome is a ribozyme. ions activate the attack of the nucleophile and stabilize the Science, 289, 878–879. departure of the 3′-oxygen. In the genomic HDV ribozyme, a 11. Collins,C.A. and Guthrie,C. (2000) The question remains: is the metal ion functions as a general base catalyst and the internal spliceosome a ribozyme? Nat. Struct. Biol., 10, 850–854. N3 moiety of a cytosine residue functions as a general acid. 12. Uhlenbeck,O.C. (1987) A small catalytic oligoribonucleotide. Nature, 328, 596–600. Hammerhead ribozymes also need two effective catalysts to 13. Haseloff,J. and Gerlach,W.L. (1988) Simple RNA enzymes with new and activate the attack by the 2′-oxygen and to stabilize the depar- highly specific endoribonuclease activities. Nature, 334, 585–591. ture of the 5′-oxygen. Metal ions are candidates for these cata- 14. Bratty,J., Chartrand,P., Ferbeyre,G. and Cedergren,R. (1993) The lysts: one works as a catalyst to assist in deprotonation of the hammerhead RNA domain, a model ribozyme. Biochim. Biophys. Acta, 1216, 345–359. nucleophile and/or the other helps in the departure of the 15. Pyle,A.M. (1993) Ribozymes: a distinct class of metalloenzymes. Science, leaving oxygen atom from the phosphorus atom (stabilization 261, 709–714. of the product). In the case of hairpin ribozymes, where catalysis 16. Yarus,M. (1993) How many catalytic RNAs? Ions and the Cheshire cat is not dependent on divalent metal ions, there must be other conjecture. FASEB J., 7, 31–39. catalysts such as a nucleobase that can assist the attack by the 17. Dahm,S.C., Derrick,W.B. and Uhlenbeck,O.C. (1993) Evidence for the role of solvated metal hydroxide in the hammerhead cleavage mechanism. 2′-oxygen and the departure of the 5′-oxygen. Biochemistry, 32, 13040–13045. In conclusion, the mechanisms of hydrolysis of RNA by 18. Steitz,T.A. and Steitz,J.A. (1993) A general two-metal-ion mechanism for ribozymes in nature are becoming clearer, and the diversity of catalytic RNA. Proc. Natl Acad. Sci. USA, 90, 6498–6502. the various candidates that might serve as catalysts is 19. Uebayashi,M., Uchimaru,T., Koguma,T., Sawata,S., Shimayama,T. and becoming apparent. Catalysts that stabilize the leaving oxygen, Taira,K. (1994) Theoretical and experimental consideration on the hammerhead ribozyme reactions – divalent magnesium-ion mediated as well as catalysts for deprotonation of the nucleophile, are cleavage of phosphorus–oxygen bonds. J. Org. Chem., 59, 7414–7420. required for effective catalytic reactions. It is noteworthy that 20. Sawata,S., Komiyama,M. and Taira,K. (1995) Kinetic evidence based on the architecture of the complex between the substrate and the solvent isotope effects for the nonexistence of a proton-transfer process in ribozyme allows perturbation of the pK of nucleobases which, reactions catalyzed by a hammerhead ribozyme – implication to the double- metal-ion mechanism of catalysis. J. Am. Chem. Soc., 117, 2357–2358. in addition to metal ions, can act as efficient catalysts (8,9,40–42). 21. Pontius,B.W., Lott,W.B. and von Hippel,P.H. (1997) Observations on The importance of nucleobases with a perturbed pK has been catalysis by hammerhead ribozymes are consistent with a two-divalent- clearly demonstrated in the case of the genomic HDV metal-ion mechanism. Proc. Natl Acad. Sci. USA, 94, 2290–2294. ribozyme (42) and more examples are being presented that 22. Zhou,D.M., Zhang,L.H. and Taira,K. (1997) Explanation by the double- metal-ion mechanism of catalysis for the differential metal ion effects on involve ribosomes (7–10). 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(1996) Ab initio investigation on nucleophilic ring opening of 1,3,2- Hirokawa Publishing Co, Tokyo, Japan. Nucleic Acids Research, 2001, Vol. 29, No. 9 1835 Figure 1. The two-dimensional structures of various ribozymes. The ribozyme or intron portion is printed in green. The substrate or exon portion is printed in black. Arrows indicate sites of cleavage by ribozymes. (A) Left, the two-dimensional structure of a hammerhead ribozyme and its substrate. Outlined letters are conserved bases that are involved in catalysis. Right, the γ-shaped structure of the hammerhead ribozyme–substrate complex. (B–F) The two-dimensional struc- tures of a hairpin ribozyme, the genomic HDV ribozyme, a group I ribozyme from Tetrahymena, a group II ribozyme from Saccharomyces cerevisiae (aiγ 5) and scherichiathe ribozyme of RNase P from E.coli, respectively. Figure 2. The two-step reaction scheme for the hydrolysis of a phosphodiester bond in RNA. First, the 2′-oxygen attacks the phosphorus atom, acting as an internal nucleophile, to generate the pentacoordinated intermediate or transtion state TS1. The 5′-oxygen then departs from the intermediate to complete cleavage at TS2. TS1 can be stabilized by a general base catalyst and TS2 can be stabilized by a general acid catalyst, as illustrated at the summits of the energy diagram.These transition states can also be stabilized by the direct binding of Lewis acids to the 2′-attacking oxygen and the 5′-leaving oxygen. Figure 3. Possible catalytic functions of metal ions in the cleavage of a phosphodiester bond. Metal ions can act as (a) a general acid catalyst, (b) a general base catalyst, (c) a Lewis acid that stabilizes the leaving group, (d) a Lewis acid that enhances the deprotonation of the attacking nucleophile and (e) an electrophilic catalyst that increases the electrophilicity of the phosphorus atom. Figure 4. A schematic representation of splicing reactions and the structures of transition states at each step. (A) The group I intron splicing reaction. (i) In the first step, the 3′-OH of the exogenous conserved G attacks the phosphorus at the 5′ splice site and generates the G-attached intron 3′–exon 2 intermediate and a free 5′ exon 1. In the second step, the 3′-OH of the 5′ exon 1 attacks the phosphorus at the 3′ splice site to produce ligated exons and the excised G-attached intron. (ii) 2+ The proposed chemical mechanism of the first step. The 3′-OH of the exogenous G is a nucleophile and the 3′-OH of the U is a leaving group. One of the Mg –1 ions [site (b)] coordinates with the 3′-OH of the G to activate the attacking group. The second [site (c)] coordinates with the 2′-OH of the G. The third [site (d)] coordinates with the pro-Sp oxygen to stabilize the transition state or the intermediate. The fourth [site (a)] coordinates with the 3′-OH of the U to stabilize the –1 leaving group. The 2′-OH also protonates the 3′-leaving oxygen of the U . It is not known whether or not the metal ion at site (d) is the same as those at the other –1 sites, (a), (b) and (c) (72). IGS represents the internal guide sequence. (B) The group II intron splicing reaction. (i) In the first step, the 2′-OH of an A residue that is conserved in the intron attacks the phosphorus at the 5′ splice site and generates an intron 3′–exon2intermediateand afree5′ exon 1. In the second step, the free 3′-OH of the 5′ exon attacks the phosphorus at the 3′ splice site to produce ligated exons and an excised intron. SER indicates the spliced-exon reopening reaction. (ii) The proposed chemical mechanisms of the first and the second steps. In the first step, the 2′-OH of an intron A residue is the nucleophile and the 3′- 2+ OH of the 5′ splice site terminus is the leaving group. One Mg ion coordinates with the 3′-OH to stabilize the leaving group. Other coordinations and/or interac- tions remain to be clarified. In the second step, the 3′-OH of the C, the 5′ splice site terminus, becomes the nucleophile and the 3′-OH of the U is the leaving group. 2+ One Mg ion coordinates directly with the 2′-OH and the 3′-OH of the U. Other coordinations and/or interactions remain to be clarified. Figure 5. (A) The mechanism of cleavage by ribonuclease A. Two imidazole residues function as general acid–base catalysts. (B) The single-metal-ion mechanism proposed for cleavage by the hammerhead ribozyme. One metal ion binds directly to the pro-Rp oxygen and functions as a general base catalyst. (C) The double- metal-ion mechanism proposed for cleavage by the hammerhead ribozyme. Two metal ions bind directly to the 2′-and 5′-oxygens. Figure 6. (A) The dependence on pH of the deuterium isotope effect in the hammerhead ribozyme-catalyzed reaction. Green circles show rate vconstants in H O; yellow circles show rate constants in D O. Solid curves are experimentally determined. The apparent plateau of cleavage rates above pH 8 is due to disruptive 2+ effects on the deprotonation of U and G residues. Dotted lines are theoretical lines calculated from pK values of hydrated Mg ions of 11.4 in H O and 12.0 in a 2 D O and on the assumption that here is no intrinsic isotope effect (α = k /k =1;where α is the coefficient of the intrinsic isotope effect). The following 2 H2O D2O (pKa(base) – pL) (pL – pKa(acid), equation was used to plot the graph of pL versus log(rate): log k = log (k )–log[1+ 10 ] – log[1 + 10 ]. In this equation, k is the rate obs max max constant in the case of all acid and base catalysts in active forms: in H O, k = k ;and in D O, k = k = k /α.(B) The isotope effects on the acidities 2 max H2O 2 max D2O H2O D2O H2O 2+ (pK – pK ) of phenols and alcohols as a function of their acid strengths (pK ). The pK of hydrated Mg ions in H O is 11.4, and the red arrow indicates a a a a 2 2+ the isotope effect of 0.65 that results in the pK of hydrated Mg ions in D O being 12.0. The pK of the N3 of cytosine in H O is 6.1 and the blue arrow indicates a 2 a 2 the isotope effect of 0.53 that results in the pK of N3 of cytosine in D O being 6.6. a 2 3+ 2+ Figure 7. Titration with Ln ions. The hammerhead ribozyme reaction was examined on a background of Mg ions. (A) Data obtained by Lott et al. (24). The proposed binding of metal ions is illustrated. (B) Data obtained by Nakamatsu et al. (161). An unmodified ribozyme (R34; red curve) and a modified ribozyme (7- 3+ deaza-R34; blue curve) were used. The rate constants were normalized by reference to the maximum rate constant ([La ]= 3 µ M). Reactions were performed under single-turnover conditions in the presence of 80 nM ribozyme and 40 nM substrate at 37°C. 2+ 2+ Figure 8. (A) Titration with Cd ions. The hammerhead ribozyme reaction was examined on a background of Ca ions. The substrate had a normal phosphate 2+ (natural substrate; blue) or an Rp-phosphorothioate group (RpS substrate; red) at the cleavage site. Solid curves indicate the rate constants for Cd -associated reactions. Dotted curves indicate the observed rate constants. (B) Thermodynamic boxes for the cleavage of the natural substrate (blue) and the RpS substrate (red) by the hammerhead ribozyme. R, ribozyme–substrate complex with all metal-binding sites occupied except the exchange site(s) examined here; M, the metal ion(s) that binds to the exchange site(s) examined here; K and K , the intrinsic dissociation constants for binding of metal ion(s) to the exchange site(s) examined dGS dTS 2+ here in the ground state and in the transition state, respectively; k , the rate of ribozyme-catalyzed cleavage with Cd ion(s) at the exchange site(s) examined cleave here; and k, the rate of non-enzymatic cleavage. The observed rate constants can be described in terms of pseudo equilibrium constant K s for the formation of the transition state (‡) from the ground state (k =[k T/h]K ,in which k is Boltzmann’s constant and h is Planck’s constant). B d B Figure 9. Reactions catalyzed by the genomic HDV ribozyme. (A) Fractions of the active species [AH] that acts as an acid catalyst (blue) and the active species [B:] that acts as a base catalyst (red), respectively. The pK of the acid catalyst is 6.1 and that of the base catalyst is 11.4 in H O. The theoretical curve for H Oin a 2 2 (B) was produced by the multiplication of these two curves. (B) Dependence on pH of the deuterium isotope effect in the HDV ribozyme-catalyzed reaction. Green circles, rate constants in H O; yellow circles, rate constants in D O; solid curves, experimental data; dotted curves, theoretical data calculated using the equation 2 2 2+ in Figure 6 and pK values for C and for hydrated Mg ions of 6.1 and 11.4 in H O and 6.5 and 12.0 in D O, respectively, assuming α = 2. The blue curve is a a 75 2 2 pH profile in 1 M NaC1 and 1 mM EDTA in the absence of divalent metal ions. (C) Energy diagram for cleavage of its substrate by an HDV ribozyme. The rate- limiting step in the reaction with the natural substrate is the cleavage of the P-(5′-O) bond. The structures of transition states TS1 and TS2 are also shown. P(V), the pentacoordinate intermediate/transition state. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nucleic Acids Research Oxford University Press http://www.deepdyve.com/lp/oxford-university-press/survey-and-summary-recent-advances-in-the-elucidation-of-the-R4gCdFeMsa

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Publisher: Oxford University Press
ISSN: 0305-1048
eISSN: 1362-4962
DOI: 10.1093/nar/29.9.1815
Publisher site: See Article on Publisher Site

Abstract

© 2001 Oxford University Press Nucleic Acids Research, 2001, Vol. 29, No. 9 1815–1834 SURVEY AND SUMMARY Recent advances in the elucidation of the mechanisms of action of ribozymes 1 1 2 1 Yasuomi Takagi , Masaki Warashina , Wojciech J. Stec , Koichi Yoshinari and 1,3, Kazunari Taira * Gene Discovery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Science City 305-8562, Japan, Polish Academy of Science, Center of Molecular and Macromolecular Studies, Department of Bioorganic Chemistry, Sienkiewicza 112, 90-363 Lodz, Poland and Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Hongo, Tokyo 113-8656, Japan Received as resubmission February 15, 2001; Revised and Accepted February 27, 2001 ABSTRACT INTRODUCTION The cleavage of RNA can be accelerated by a number Naturally existing catalytic RNAs include hammerhead, hairpin, hepatitis delta virus (HDV) and Varkud Satellite (VS) of factors. These factors include an acidic group ribozymes; group I and II introns; and the RNA subunit of (Lewis acid) or a basic group that aids in the deprotona- RNase P (1–6). The structures of these catalytic RNAs are tion of the attacking nucleophile, in effect enhancing shown in Figure 1. In addition, recent structural and chemical the nucleophilicity of the nucleophile; an acidic analyses strongly suggest that the ribosomal RNA is a group that can neutralize and stabilize the leaving ribozyme (7–10) and the possibility that the RNA component group; and any environment that can stabilize the of the spliceosome might also be a ribozyme (11). pentavalent species that is either a transition state or Extensive efforts over the 15 years that followed the discovery of ribozymes (1,2) have revealed details of the a short-lived intermediate. The catalytic properties of mechanisms of the ribozyme-mediated cleavage (or ligation) ribozymes are due to factors that are derived from of RNA. Ribozymes have been considered to be ‘fossil mole- the complicated and specific structure of the cules’ that originated in a hypothetical prebiotic RNA world ribozyme–substrate complex. It was postulated and it is likely that elucidation of their mechanisms of action initially that nature had adopted a rather narrowly will enhance our understanding of the life processes of primi- defined mechanism for the cleavage of RNA. tive organisms (12–33). Since the earliest research on However, recent findings have clearly demonstrated ribozymes, it was assumed that all ribozymes are metalloen- the diversity of the mechanisms of ribozyme-catalyzed zymes that require divalent metal ions for catalysis and that all must operate by a basically similar mechanism. However, reactions. Such mechanisms include the metal- recent advances have revealed examples of cleavage by hairpin independent cleavage that occurs in reactions ribozymes that are independent of divalent metal ions (34–39). catalyzed by hairpin ribozymes and the general Thus, the various types of ribozyme appear to exploit different double-metal-ion mechanism of catalysis in reactions cleavage mechanisms, which depend upon the architecture of catalyzed by the Tetrahymena group I ribozyme. the individual ribozyme. Furthermore, it was proposed recently Furthermore, the architecture of the complex between that nucleobases in the HDV ribozyme might be candidates for the substrate and the hepatitis delta virus ribozyme participants in acid/base catalysis (40–42). allows perturbation of the pK of ring nitrogens of cyto- In addition, even hammerhead ribozymes, generally charac- terized as typical metalloenzymes, can no longer be unambig- sine and adenine. The resultant perturbed ring nitro- uously categorized (43,44). Recent findings indicate that the gens appear to be directly involved in acid/base hammerhead ribozyme might operate via a variety of cleavage catalysis. Moreover, while high concentrations of mechanisms, depending on the conditions of the reaction. monovalent metal ions or polyamines can facilitate Nevertheless, there is no doubt that RNA catalysts with groups cleavage by hammerhead ribozymes, divalent metal that are poorly functional under physiological conditions do ions are the most effective acid/base catalysts under cooperate with metal ions to exert their catalytic activity and physiological conditions. that many ribozymes can exploit divalent metal ions as cofactors *To whom correspondence should be addressed at: Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Hongo, Tokyo 113-8656, Japan. Tel: +81 35841 8828; Fax: +81 298 61 3019; Email: [email protected] The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors 1816 Nucleic Acids Research, 2001, Vol. 29, No. 9 Figure 1. The two-dimensional structures of various ribozymes. The ribozyme or intron portion is printed in green. The substrate or exon portion is printed in black. Arrows indicate sites of cleavage by ribozymes. (A) Left, the two-dimensional structure of a hammerhead ribozyme and its substrate. Outlined letters are conserved bases that are involved in catalysis. Right, the γ-shaped structure of the hammerhead ribozyme–substrate complex. (B–F) The two-dimensional struc- tures of a hairpin ribozyme, the genomic HDV ribozyme, a group I ribozyme from Tetrahymena, a group II ribozyme from Saccharomyces cerevisiae (aiγ 5) and the ribozyme of RNase P from Escherichia coli, respectively. and as stabilizers of their respective higher-order structures. considered to be roughly equivalent to the non-enzymatic The widespread potential utility of RNA molecules as catalysts hydrolysis of RNA, with inversion of the configuration at a and the events during reactions catalyzed by ribozymes, in phosphorus atom suggesting a direct in-line attack with devel- particular the actions of catalytic functional groups such as opment of a pentacoordinate transition state or intermediate. metal ions and pK -perturbed nucleobases, have generated The chemical cleavage requires two events, which can occur considerable interest (1–49). In this article we shall review either via a two-step mechanism or via a concerted mechanism various naturally existing ribozymes that cleave RNAs, (4,5,25,45). focusing mainly on the various mechanisms of catalysis. In the first step of the non-enzymatic hydrolysis of RNA (25,50–52), the 2′-OH attacks the adjacent scissile phosphate, acting as an internal nucleophile (transition state 1; TS1) (Fig. 2). CLEAVAGE OF THE PHOSPHODIESTER BOND In the second step, the 5′-oxygen of the leaving nucleotide is For the cleavage of RNA phosphodiester linkages, three types released to produce a 3′-end 2′,3′-cyclic phosphate and a of large ribozyme, namely, group I and II introns and the catalytic 5′-OH terminus (transition state 2; TS2). Of the two putative RNA subunit of RNase P, accept external nucleophiles (the 2′- transition states, TS2 is the overall rate-limiting state OH group of an internal adenosine in the case of the group II [i.e., attack by the 2′-OH on the phosphorus atom is easier than intron). By contrast, small ribozymes, such as hammerheads, cleavage of the P-O(5′) bond and, thus, TS2 always has higher hairpins, HDV and the VS ribozyme, use an internal nucleophile, energy than TS1] (25). This conclusion was confirmed in namely, the 2′-oxygen of the ribose moiety at the cleavage site, experiments with an RNA analog with a 5′-mercapto leaving with resultant formation of a 3′-terminal 2′,3′-cyclic phos- group. If the formation of the intermediate were the rate- phate. In general, ribozymes catalyze the endonucleolytic limiting step (i.e., if TS1 were a higher-energy state than TS2) transesterification of the phosphodiester bond, requiring struc- in the natural RNA, the phosphorothiolate RNA (RNA with a tural and/or catalytic divalent metal ions under physiological 5′-bridging phosphorothiolate at the scissile linkage) should be conditions. The reactions catalyzed by small ribozymes are hydrolyzed at a rate similar to the rate of the hydrolysis of the Nucleic Acids Research, 2001, Vol. 29, No. 9 1817 Figure 3. Possible catalytic functions of metal ions in the cleavage of a phos- phodiester bond. Metal ions can act as (a) a general acid catalyst, (b) a general base catalyst, (c) a Lewis acid that stabilizes the leaving group, (d)aLewis acid that enhances the deprotonation of the attacking nucleophile and (e)an electrophilic catalyst that increases the electrophilicity of the phosphorus atom. natively, hydrogen bonding between a metal-bound water molecule and the non-bridging oxygen might stabilize the Figure 2. The two-step reaction scheme for the hydrolysis of a phosphodiester charged trigonal-bipyramidal intermediate (or transition state). bond in RNA. First, the 2′-oxygen attacks the phosphorus atom, acting as an internal nucleophile, to generate the pentacoordinated intermediate or transi- Metal ions can function in several different ways as cofactors in tion state, TS1. The 5′-oxygen then departs from the intermediate to complete ribozyme-catalyzed reactions, as described above, and cleavage at TS2. TS1 can be stabilized by a general base catalyst and TS2 can proposed mechanisms for the reactions catalyzed by several be stabilized by a general acid catalyst, as illustrated at the summits of the energy diagram. These transition states can also be stabilized by the direct ribozymes have taken advantage of such functions. Moreover, binding of Lewis acids to the 2′-attacking oxygen and the 5′-leaving oxygen. it is difficult to imagine that a specific ribozyme might exploit multiple mechanisms (for example, coexistence in a reaction of the left structure and the central structure in Figure 3 at the natural RNA because the 5′-bridging phosphorothiolate transition state of a ribozyme-catalyzed reaction) under a linkage would not be expected to enhance the attack by the single set of physiological conditions. Significant aspects of 2′-oxygen (53). By contrast, if the decomposition of the inter- these functions of metal ions might be subsumed by nucleo- mediate were the rate-limiting step (i.e., if TS2 were a higher- bases if their pK values could be adjusted appropriately. The energy state than TS1) in the natural RNA, the phosphoro- full details of the mechanisms of action of metalloenzymes thiolate RNA would be expected to be hydrolyzed much more remain to be elucidated. rapidly than the natural RNA because the pK of a thiol is >5 units lower than that of the corresponding alcohol. Several groups have confirmed that the phosphorothiolate RNA is LARGERIBOZYMES significantly more reactive than the corresponding natural The group I intron, the group II intron and the RNA subunit of RNA in non-enzymatic hydrolytic reactions (25,45,54–56) RNase P are categorized as large ribozymes. Group I and II and, thus, TS2 is, indeed, always a higher-energy state than introns are found in bacteria and in the organelles of higher TS1. plants, fungi and algae (57,58). These introns are spliced out of their primary transcripts by a two-step mechanism [Fig. 4A(i) and POSSIBLE CATALYTIC FUNCTIONS OF METAL B(i)]. In the first step of splicing, the 5′ splice site is attacked IONS IN THE CLEAVAGE OF RNA by the 3′-OH of the external guanosine (group I). Alter- natively, it is attacked by the 2′-OH of the internal adenosine If ribozymes operate as metalloenzymes (4,5,15–25,27,45), the residue or by a hydroxide ion, in the case of hydrolysis (group possible catalytic functions of metal ions can be summarized II).Inthe second step,the 3′-OH of the 3′-end of the upstream as follows (Fig. 3). exon attacks the 3′ splice site to produce splicing products. · A metal-coordinated hydroxide ion might act as a general RNase P is an endonuclease that generates the mature base, abstracting the proton from the 2′-OH (Fig. 3b) or, alter- 5′-ends of tRNAs. In bacterial RNase P, the RNA subunit natively, a metal ion might act as a Lewis acid to accelerate the (RNase P ribozyme) has catalytic activity and the protein deprotonation of 2′-OH by coordinating directly with the 2′- component is thought to act only to facilitate the binding of the oxygen (Fig. 3d). anionic RNase P ribozyme to its substrate. However, mutations · The developing negative charge on the 5′-oxygen leaving in either the gene for the RNA or the gene for the protein can group might be stabilized by a proton that is provided by a inactivate RNase P in vivo, demonstrating that both compo- solvent water molecule or by a metal-bound water molecule as a general acid catalyst (Fig. 3a) or, alternatively, by direct nents are necessary for natural enzymatic activity. In the cleavage by the RNase P ribozyme, a scissile-site phosphate is coordination of a metal ion that acts as a Lewis acid catalyst attacked by a hydroxide ion to leave a 3′-oxygen and to (Fig. 3c). produce a 5′-phosphate terminus. · Direct coordination of a metal ion to the non-bridging oxygen might render the phosphorus center more susceptible All of these ribozyme reactions proceed with inversion of to nucleophilic attack (electrophilic catalysis; Fig. 3e) or, alter- configuration at a phosphorus atom (59–62), suggesting direct 1818 Nucleic Acids Research, 2001, Vol. 29, No. 9 Figure 4. A schematic representation of splicing reactions and the structures of transition states at each step. (A) The group I intron splicing reaction. (i) In the first step, the 3′-OH of the exogenous conserved G attacks the phosphorus at the 5′ splice site and generates the G-attached intron 3′–exon 2 intermediate and a free 5′ exon 1. In the second step, the 3′-OH of the 5′ exon 1 attacks the phosphorus at the 3′ splice site to produce ligated exons and the excised G-attached intron. (ii) 2+ The proposed chemical mechanism of the first step. The 3′-OH of the exogenous G is a nucleophile and the 3′-OH of the U is a leaving group. One of the Mg –1 ions [site (b)] coordinates with the 3′-OH of the G to activate the attacking group. The second [site (c)] coordinates with the 2′-OH of the G. The third [site (d)] coordinates with the pro-Sp oxygen to stabilize the transition state or the intermediate. The fourth [site (a)] coordinates with the 3′-OH of the U to stabilize the –1 leaving group. The 2′-OH also protonates the 3′-leaving oxygen of the U . It is not known whether or not the metal ion at site (d) is the same as those at the other –1 sites, (a), (b) and (c) (72). IGS represents the internal guide sequence. (B) The group II intron splicing reaction. (i) In the first step, the 2′-OH of an A residue that is conserved in the intron attacks the phosphorus at the 5′ splice site and generates an intron 3′–exon 2intermediateand afree5′ exon 1. In the second step, the free 3′-OH of the 5′ exon attacks the phosphorus at the 3′ splice site to produce ligated exons and an excised intron. SER indicates the spliced-exon reopening reaction. (ii) The proposed chemical mechanisms of the first and the second steps. In the first step, the 2′-OH of an intron A residue is the nucleophile and the 3′-OH 2+ of the 5′ splice site terminus is the leaving group. One Mg ion coordinates with the 3′-OH to stabilize the leaving group. Other coordinations and/or interactions remain to be clarified. In the second step, the 3′-OH of the C, the 5′ splice site terminus, becomes the nucleophile and the 3′-OH of the U is the leaving group. One 2+ Mg ion coordinates directly with the 2′-OH and the 3′-OH of the U. Other coordinations and/or interactions remain to be clarified. 2+ in-line attack with development of a pentacoordinate transition rule, a ‘hard acid’, such as a Mg ion, prefers to bind to a ‘hard state or intermediate (59–72). base’ oxygen atom rather than to a ‘soft base’ sulfur atom. By 2+ 2+ contrast, a ‘soft acid’, such as Cd or Zn ions, prefers to bind 2+ to a ‘soft base’ sulfur atom. A Mn ionis alsosofterthana THE MECHANISM OF REACTIONS CATALYZED BY 2+ Mg ion and, thus, the former can bind to a soft sulfur atom (as THE GROUP IINTRONRIBOZYME 2+ well as to a hard oxygen atom). This ability of Mn ions is believed to be the origin of the manganese rescue effect. In studies of the reactions mediated by the ribozyme from the Tetrahymena group I intron, detailed kinetic and thermo- Analysis of both the thio effect and of soft acid rescue 2+ 2+ 2+ dynamic analysis, combined with modifications at the atomic effects, such as the rescue effects of Cd ,Mn and Zn ions, level, helped to define the reaction mechanism of this has contributed significantly to our understanding of the catalytic ribozyme at the atomic level (18,63,66,68–72). Modification at mechanism of the first step of the reaction catalyzed by the the atomic level has generally involved replacement by a sulfur group I intron. Such analysis has revealed the importance of atom of an oxygen atom that has the potential to interact with a three to four independent metal ions, as shown in Figure 4A(ii) catalytically important metal ion. The observed reduction in (69,71). It is generally accepted that the group I intron is a 2+ the cleavage rate in the presence of Mg ions after such metalloenzyme that operates via the general double-metal-ion 2+ modification (the ‘thio effect’) and the observed restoration of mechanism of catalysis, in which a Mg ion at site (b) [see 2+ a normal cleavage rate in the presence of Mn ions (the Fig. 4A(ii) for locations of (a), (b), (c) and (d)] enhances the ‘manganese rescue effect’) have been taken as evidence that deprotonation of the 3′-OH of the guanosine nucleophile and a 2+ supports the direct coordination of the atom in question with a Mg ion at site (a) stabilizes the leaving 3′-bridging oxygen of metal ion. This phenomenon can be explained by the HSAB U in the transition state. In this case, the divalent metal ions –1 (Hardand Soft, Acidand Base)rule(73,74).Accordingtothis function as Lewis acids for activation of the nucleophile and Nucleic Acids Research, 2001, Vol. 29, No. 9 1819 stabilization of the leaving group by coordinating directly with result suggests that a metal ion at site (c) coordinates directly them (63). This mechanism corresponds, in the reactions cata- with the 2′-OH. lyzed by small ribozymes, to the central mechanism shown in In addition to the coordination of metal ions discussed Figure 3 with the stabilization of both TS1 and TS2 (Fig. 2) by above, other interesting interactions have been proposed. two metal ions. Linear free-energy analysis of the cleavage of oligonucleotide These details of coordination at the catalytic site were substrates with a series of 2′-substituents at U indicated that –1 derived from the following observations. The substitution of theeffect on therateof the 2′-OH group is larger than might be the 3′-oxygen of the guanosine nucleophile with a sulfur atom expected from simple inductive effects (81). The weaker reduced the rate of the reverse reaction in the presence of the electron-withdrawing 2′-OH enhanced the chemical cleavage 2+ hard acid, namely, Mg ions, and an efficient cleavage was step to a greater extent than did the more strongly electron- 2+ 2+ restored by Mn ions (66). This result suggests that a Mg ion withdrawing 2′-F atom of the corresponding 2′-deoxy-2′-fluoro at site (b) coordinates with the 3′-oxygen of the guanosine derivative. Therefore, the possibility was recently examined of a nucleophile to activate the first step. Next, the 3′-bridging symmetrical transition state, in which the 2′-OH of U might –1 phosphorothiolate substrate (3′-S substrate), in which the or might not interact with a metal ion [as observed at site (c) in 3′ leaving oxygen had been replaced by a sulfur atom, had a Fig. 4A(ii)] (71). Despite the absence of lone-pair electrons at dramatically reduced cleavage rate for the forward reaction in the 2′-NH group that need to interact with a metal ion, the 2+ thepresenceofMg ions. An efficient cleavage was restored + higher reactivity of the substrate with a 2′-deoxy-2′-NH 2+ 2+ by Mn ions (63,75). This result suggests that a Mg ion at group than that of the substrate with a 2′-OH group at U –1 site (a) in Figure 4A(ii) coordinates with the 3′-leaving oxygen suggested that interaction of a metal ion with the 2′-OH of U –1 during cleavage. These observations can be explained by the might not be important for catalysis by the group I intron 2+ double-metal-ion model, in which one Mg ion coordinates + ribozyme. The higher reactivity of the 2′-NH derivative with the nucleophile to activate the attacking group and the suggests that donation of a hydrogen bond from the 2′-group to 2+ other Mg ion coordinates with the 3′-leavingoxygentostabi- the neighboring 3′-leaving oxygen might allow specific stabiliza- lize the developing negative charge during RNA cleavage. tion of the transition state relative to the ground state, thereby 2+ The possibility of coordination of a Mg ion at site (d) in facilitating the chemical cleavage step. Figure 4A(ii) with the pro-Sp oxygen was suggested on the The 2′-OH of U ,the 2′-OH of A and the exocyclic amino –1 207 basis of the following experimental data. The RpS substrate, in group of G have been referred to as a catalytic triad (70). which the pro-Rp oxygen at the scissile phosphate had been However, the observation that the chemical cleavage step with replaced by sulfur, was cleaved at a modestly reduced rate a2′-NH derivative is faster than that with the substrate with a (76). By contrast, the SpS substrate, in which the pro-Sp 2′-OH (the natural substrate), despite the absence of lone-pair oxygen at the scissile phosphate had been replaced by sulfur, electrons at the 2′-NH group that can accept a hydrogen bond 2+ 3 had a drastically reduced cleavage rate in the presence of Mg from A -OH, suggests another possibility for the arrange- ions (71,75,77). Furthermore, the SpS/3′-S substrate, in which ment of active-site groups within this network of interactions not only the pro-Spoxygen but alsothe 3′-leaving oxygen had (71,72). been replaced by sulfur atoms at the same scissile phosphate, Even though the ribozyme-mediated chemical cleavage step was cleaved at a lower rate than the 3′-S substrate in the with the 2′-OH group at U (the natural substrate) is signifi- 2+ 2+ –1 presence of Mn ions on a background of Mg ions. An cantly (1000-fold) faster than that with 2′-H, with the metal- efficient cleavage of the SpS/3′-S substrate, with the double- 2+ 2+ 2+ binding site (a) in Figure 4A(ii) being occupied by a Mn ion, thio substitution, was restored by Zn or Cd ions, which are 2+ 2+ the rate constants for reactions with the 3′-S substrates are more thiophilic than Mn ions, on a background of Mg ions similar, irrespective of whether there is a 2′-OH or 2′-H at U . –1 (75). Thus, a thio effect seemed apparent at the pro-Sp oxygen, 2+ 2+ 2+ Moreover, in the presence of Mg ions, with the metal-binding and rescue both by Cd andbyZn ions was also evident. 2+ site (a) being unoccupied by a metal ion, the rate constants for These results suggested that a Mg ion(s) might coordinate reactions with the 3′-S substrates are similar with a 2′-OH or with the pro-Sp oxygen, as well as with the 3′-leaving oxygen. with a 2′-H at U , indicating that the 2′-OH at U does not –1 –1 However, in consideration of the above results, we should contribute significantly to the chemical cleavage of the note that an efficient cleavage of the SpS substrate, with phosphorus–sulfur bond with the 3′-mercapto leaving group. the single-thio substitution, could not be restored either by 2+ 2+ 2+ Since sulfur is a weaker acceptor of a hydrogen bond than is Mn ions or by Zn or Cd ions (75). This observation 2+ oxygen and, furthermore, since sulfur is a significantly better prevents us from ruling out the possibility that a Mg ion does leaving group than oxygen, the 3′-mercapto leaving group not coordinate with the pro-Sp oxygen in a direct manner suppresses the catalytic advantage provided by a hydrogen during the first step in cleavage by the group I intron ribozyme bond from the 2′-OH in the native transition state (71). (72). Further investigations are needed to determine whether 2+ The second step of the splicing reaction is catalyzed within direct coordination of a Mg ion occurs at site (d). the same catalytic site as the first step (18,82,83). Moreover, in Additional coordination has also been proposed at the 2+ 2+ thepresenceofMg ions, both the reverse reaction of the first catalytic site. A Mg ion at site (c) in Figure 4A(ii) might step and the forward reaction of the second step were inhibited interact directly with the 2′-OH of the guanosine, as suggested with the RpS substrate (note that the pro-Rpoxygenat these by experiments with a 2′-amino-2′-deoxyguanosine substrate and various metal ions in the ribozyme reaction (78–80). The steps corresponds to the pro-Sp oxygen in the forward reaction cleavage rate was reduced by replacement of the 2′-OH with of the first step). These observations indicate that the stereo- 2+ 2′-NH on a background of Mg ions. An efficient cleavage chemical requirements are the same in both reactions 2+ 2+ was restored by addition of soft Mn or Zn ions (78,79). This (60,63,76,84). Therefore, the mechanism of the second step is 1820 Nucleic Acids Research, 2001, Vol. 29, No. 9 2+ considered to be analogous to the mechanism of the first step cleaved faster in the presence of Mn ions than in the presence 2+ (18). of Mg ions at higher pH (at higher pH, the 2′-amino group It is clear that the analysis of thio effects, rescue experiments exists in a neutral form, as -NH , and not in the protonated + 2+ form, -NH )sinceaMn ion binds to the 2′-NH group better and other experiments with derivatives have contributed 3 2 2+ than a Mg ion (nitrogen is a softer base than oxygen and the significantly to our understanding of the mechanism of action 2+ 2+ of the large group I intron ribozyme of Tetrahymena.All the Mn ion is a softer acid than the Mg ion). This interaction with the 2′-oxygen at the second step involves a single metal available data appear to support the refined double-metal-ion ion in the transition state, as indicated by the fact that the mechanism of catalysis (18) that is shown in Figure 4A(ii). 2+ dependence on the concentration of Mn ions with the 2′- THE GROUP II INTRON RIBOZYME 2+ amino substrate on a background of Mg ions is consistent with a single-metal-ion exchange (85). In the splicing reactions catalyzed by the group II intron ribozyme (Fig. 4B), the first step was blocked when the RpS The 3′-S substrate reduced the cleavage rate of the second 2+ substrate was used (61) and a small thio effect was observed step by ∼100-fold in the presence of Mg ions, and the reac- 2+ 2+ with the SpS substrate (64), suggesting that the pro-Rpoxygen tion was rescued completely by the addition of Mn ,Co or 2+ makes an essential interaction in the transition state and that Cd ions in the tripartite assay (67,85). However, this the pro-Sp oxygen is also involved in some kind of interaction. substrate had no effect on the bimolecular cis-splicing assay in No cleavage of the 3′-S substrate was observed in the presence which the rate-limiting step appeared to be the conformational 2+ 2+ 2+ 2+ of Mg ions but the reaction was rescued by Mn ,Zn or rearrangement (67). This result indicates that Mg ions also 2+ 2+ Cd ions (67). This result indicates that a Mg ion acted to acted in the second step to stabilize the 3′-leavingoxygenby stabilize the 3′-leaving oxygen by direct coordination. direct coordination, as was the case in the first step. This inter- 2+ The second step was monitored by a tripartite assay (trans- action with the 3′-oxygen also involved a single Mn ion in the splicing), in which an oligonucleotide that corresponded to the transition state, as observed for the cleavage of the 2′-amino 3′-splice site was added after the formation of the ribozyme/ substrate (85). We should emphasize, however, that the prefer- 5′-exon RNA complex, because the second step might be ences of the 3′-S substrate for metal ions differed between the 2+ 2+ 2+ 2+ 2+ masked by the rate-limiting conformational rearrangement first step (Mn ,Zn or Cd ) and the second step (Mn ,Co 2+ between the first step and the second step that was observed in or Cd ), indicating the differences between the environments the bimolecular assay (cis-splicing). In contrast to the results of of metal ions at the two different transition states for each analysis of the first step, the cleavage of the RpS substrate was splicing step. strongly inhibited but the SpS substrate had essentially no The moiety that activates the attacking nucleophile in the inhibitory effect (85). The actual stereospecificity for the thio two independent splice steps of the reaction of the group II substitution is reversed between the first step and the second intron ribozyme remains to be identified but it is apparent that 2+ step (both steps were inhibited in the reaction with the RpS aMg ion binds to the leaving 3′-oxygen to stabilize the tran- substrate; note that the pro-Rpoxygeninthe forwardreaction sition state at each step. of the first step corresponds to the pro-Sp oxygen in the reverse reaction of the first step and in the forward reaction of the THE RNASE P RIBOZYME second step). Thus, the second step is not the reversal of the first step, unlike results for the group I intron ribozyme. Even In reactions catalyzed by the RNA subunit of bacterial RNase 2+ though the pro-Rp oxygen atom appears to make an essential P, there is a requirement for both divalent cations (e.g., Mg or 2+ + + interaction in the transition state, the nature of this interaction Mn ) and monovalent cations (e.g., K or NH ) (62). Mono- has not been defined since no rescue by thiophilic metal ions valent cations appear to be involved in the stabilization of the can be observed. structure during the cleavage reaction in the absence of The group II intron ribozyme can also hydrolyze the bond proteins in vitro. By contrast, divalent metal cations are between spliced exons [the spliced-exon reopening (SER) required for the chemical cleavage itself, and not only for reaction, which corresponds to the reverse reaction of the structural stabilization. During the chemical cleavage, a second step]. This SER reaction proceeds with the RpS hydroxide ion, activated by metal ions, is thought to act as a substrate but not with the SpS substrate (64). Since this step nucleophile (86,87). Though the details of the reaction mecha- would be expected to be blocked with the RpS substrate if it nism are not fully understood, it has been proposed that three 2+ followed the same reaction pathway as that of the first step of Mg ions participate in the transition state, because the slope the splicing reaction, the observed SER reaction supports the of the Hill plot for the cleavage rate versus the concentration 2+ 2+ conclusion that the second step is not the reversal of the first of Mg ions was 3.2, and that one of the catalytic Mg ions step, as mentioned above. coordinates directly to the pro-Sp oxygen at the scissile phosphate. The substitution of the 2′-OH of the leaving ribonucleoside 2′-Deoxy substitution at the cleavage site reduced the apparent 2+ (the U residue of the intron in Fig. 4B) at the 3′-splice site with number of bound Mg ions and decreased the apparent affinity 2+ a hydrogen atom reduced the rate of the second step ∼700-fold for Mg ions, suggesting that the 2′-oxygen might be one of 2+ (85). Moreover, even though substitution with a methoxy the Mg -binding sites. Furthermore, 2′-methoxy substitution group or a fluorine atom, respectively, reduced the rate simi- at the cleavage site decreased the cleavage rate, suggesting that larly to or significantly more than substitution with a hydrogen the 2′-OH might be involved in stabilizing the 3′-leaving atom, substitution with an amino group resulted in a rate that oxygen as the donor of a hydrogen bond (87). was ∼10-fold higher than that with a hydrogen atom (85). According to a recent report, the cleavage rate of the RpS 2+ These results suggest that the ability to donate a hydrogen bond substrate in the presence of Mg ions was at least 1000-fold from the 2′-OH group is important. The 2′-amino substrate was lower than the cleavage rate of the natural substrate. The Nucleic Acids Research, 2001, Vol. 29, No. 9 1821 reduction was, however, rescued by thiophilic metal ions, such certain pH between the ∆ pK values of metal ions in water and 2+ 2+ 2+ as Cd and Mn ions (background Mg ions are needed for the difference in the observed rates of cleavage in the presence rescue in the case of RNase P ribozyme from Bacillus subtilis), of the corresponding metal ions suggested a single-metal-ion 2+ suggesting the direct coordination of a metal ion to the pro-Rp mechanism in which Mg -hydroxide acts as a general base 2+ oxygen (88,89). Since the Hill coefficient for Cd rescue was catalyst (17). However, it was also noted that a general double- 1.8, it was proposed that two metal ions coordinate to the metal-ion mechanism, in which metal ions act as Lewis acids pro-Rp oxygen in a modified model, which is consistent with that coordinate directly to the 2′-OH and the 5′-leaving the two-metal-ion model of Steitz and Steitz (18). By contrast, oxygen, for activation of a nucleophile and for stabilization of the cleavage reaction was also blocked with the SpS substrate a developing negative charge on the leaving group, respec- (with reduction of the binding affinity of the substrate for the tively, might also explain reactions catalyzed by hammerhead ribozyme in the ground state in the case of the RNase ribozymes (20–22,24,25). Pribozyme from Escherichia coli) and the cleavage site was It should also be noted that, under extreme conditions (in the + + shifted in the 5’ direction. The reduced rate of cleavage of the presence of 1–4 M monovalent cations, such as Li ,Na and 2+ 2+ SpS substrate was not enhanced by Cd and Mn ions, an NH ), hammerhead ribozymes do not require divalent metal observation that suggests the possibility of a crucial role for the ions for catalysis (43). On the basis of this observation, some pro-Sp oxygen in stabilization of the transition state or that researchers have claimed that hammerhead ribozymes are not might be attributable to the steric exclusion of catalytic metal metalloenzymes (see below). ions (88,89). Similar effects of RpS and SpS substrates were HDV ribozymes are derived from the genomic and the anti- also observed with the eukaryotic nuclear RNase P ribozyme, genomic RNAs of hepatitis delta virus (105–108). In studies of in which the RNA is thought to be the catalytic component and reactions catalyzed by HDV ribozymes, three groups demon- to be evolutionarily related to the bacterial RNase P ribozyme strated recently that an intramolecular functional group, (90). However, no thio effect was observed in the case of namely N3 at C in the antigenomic HDV ribozyme and N3 at RNase P from plant chloroplasts, whose catalytic component C in the genomic HDV ribozyme, can, in fact, act as a true appears to be a protein (91). catalyst (40–42). However, with respect to the roles of these The 3′-S substrate also prevented cleavage at the correct site N3s, two different mechanisms, namely general base catalysis by the bacterial RNase P ribozyme and the cleavage site was and general acid catalysis, were proposed. In the former moved to the next unmodified phosphodiester bond in the scenario, it was proposed that the deprotonated N3 of C 5′-direction completely. The reduction in the cleavage rate was might be involved in cleavage as a general base that abstracts a 2+ 2+ not rescued by thiophilic Cd or Mn ions (92). While the proton from the 2′-OH to promote its nucleophilic attack on the absence of rescue by thiophilic metal ions does not reveal the scissile phosphate in the transition state of reactions catalyzed molecular nature of the inhibitory effects (the thio effect and by the antigenomic HDV ribozyme (41). In the latter case, it 2+ shifting of the cleavage site), prevention of binding of a Mg was proposed that the protonated N3 of C in the genomic ion to the 3′-leaving atom as a result of the thio substitution HDV ribozyme might act as a general acid to stabilize the provides one possible explanation. In addition, it is possible developing negative charge at the 5′-leaving oxygen and that a that several chemical and structural changes occur upon the metal ion might act as a general base (42). However, although introduction of a bulky sulfur atom. further investigations are required, there remains the possi- bility that the catalytic mechanism of the antigenomic ribozyme is the same as that of the genomic ribozyme (109). SMALL RIBOZYMES In discussion of the reaction catalyzed by the genomic HDV Hammerhead, HDV, hairpin and VS ribozymes are categorized as ribozyme, the importance has been emphasized of the neutraliza- small ribozymes because they are smaller than the ribozymes tion of the substantial negative charge that develops on the discussed above. Each of these naturally existing ribozymes 5′-leaving oxygen and the essential role of general acid catalyzes the endonucleotic cleavage of RNA via a mechanism catalysis in the cleavage of RNA (42). Such issues should be that involves nucleophilic attack by a 2′-OH group on the phos- relevant not only in the case of reactions catalyzed by HDV phorus of the neighboring phosphodiester bond, generating 5′-OH ribozymes but also in the case of reactions catalyzed by other and 2′,3′-cyclic phosphate termini (for reviews, see 4,25,93). small ribozymes since cleavage of the bond between phos- The cleavage reactions catalyzed by these ribozymes appear to phorus and the 5′-oxygen is the overall rate-limiting step, as proceed with inversion of the configuration at the phosphorus shown in Figure 2 (see below; 22,25,56). The efficient atom suggesting a direct in-line attack with development of a cleavage of a phosphodiester bond requires both the activation pentacoordinate transition state or intermediate (42,94–97). of the 2′-attacking oxygen and the stabilization of the The smallest of the naturally occurring catalytic RNAs that 5′-leaving oxygen. have been identified to date are the hammerhead ribozymes Hairpin ribozymes were originally derived from the minus (Fig. 1A), which were found in several plant viral satellite strand of the satellite RNA of tobacco ringspot virus (sTRSV), RNAs, a viroid RNA and the transcript of a nuclear satellite chicory yellow mottle virus type 1 (sCYMV1) and arabis DNA of a newt (98; for reviews, see 3,4). These ribozymes mosaic virus (sArMV) (110–113). Hairpin and hammerhead have been extensively investigated, in particular with respect ribozymes can also catalyze the ligation of cleaved products, to the mechanism of action of catalytic metal ions with the ligation efficiency being much higher for the hairpin (17,25,27,45,46). ribozyme than the hammerhead. The ligation reaction is Hammerhead ribozymes have a basic requirement for divalent thought to be the reverse of the cleavage reaction since it uses 2+ metal ions, such as Mg ions (5,6,17–22,24,25,27,45,99–104). the same termini as those produced upon cleavage. Hairpin In studies of the hammerhead reaction, the relationship at a ribozymes favor the ligation reaction rather than cleavage 1822 Nucleic Acids Research, 2001, Vol. 29, No. 9 (ligation occurs 10-fold faster than cleavage). By contrast, chemical cross-linking studies (Fig. 1A, right) (26,139–142). hammerhead ribozymes favor the cleavage reaction [cleavage Such studies indicate the involvement of two reversed-Hoogs- occurs ≥100-fold faster than ligation (47,114–116)]. The ratio teen G·A base pairs between G ·A and A ·G , aswellasa non- 8 13 9 12 of equilibrium constants (k /k ) can be explained by Watson–Crick A ·U base pair that is formed by a single cleavage ligation 14 7 the differences between entropies: the loss of entropy that hydrogen bond. These base pairs are followed by stem II and are occurs with ligation is smaller for the hairpin than for the stacked ‘coaxially’ onto the non-Watson–Crick A ·U base 15.1 16.1 hammerhead ribozyme, indicating that the more rigid hairpin pair, with resultant formation of a pseudo-A-form helix by structure undergoes a smaller change in dynamics on ligation stems II and III. Four sequential nucleotides (C U G A )form 3 4 5 6 than the more flexible hammerhead (117). Catalysis by hairpin a ‘uridine-turn’ motif, allowing the phosphate backbone to turn ribozymes in the absence of metal ions has been reported by and connect with stem I. The uridine-turn forms a catalytic several groups independently (34–39). Hairpin ribozymes can pocket into which the nucleobase at the cleavage site, namely be considered to be a distinct class of ribozymes that do not C , is inserted (133). The crystal structure of the enzyme– require metal ions as cofactors (118). The catalyst(s) seems to product complex of the hammerhead ribozyme has been deter- be a nucleobase(s). mined (137). The structure suggests that the distance between C and G /A in the transition state is smaller than previously The VS ribozyme originated from the mitochondria of 17 5 6 proposed and that dramatic conformational changes, which certain isolates of Neurospora (119). The reaction catalyzed by 2+ include C , occur in the pathway from the ground state to the the VS ribozyme requires a divalent cation such as the Mg transition state. ion (120). Some regions that are important for catalysis and some interactions between the phosphate backbone and metal Structural metal ions in reactions catalyzed by ions have been identified (121–123). The reaction appears to hammerhead ribozymes be independent of pH but the possibility exists for a conforma- tional change prior to cleavage that might mask a dependence It is generally accepted that the tertiary structures of RNA on pH (120,124). The catalytic group(s) in the cleavage molecules are stabilized by metal ions. The roles of metal ions reaction has not yet been unambiguously identified. in ribozyme-catalyzed reactions are of two distinct types: metal ions can act as catalysts during the chemical cleavage step, as shown in Figure 3; and they can also stabilize the REACTIONS CATALYZED BY HAMMERHEAD conformation of the ribozyme–substrate complex. The ion- RIBOZYMES dependent changes in the conformation of a hammerhead ribozyme can be easily followed by monitoring the influence Hammerhead ribozymes are among the smallest catalytic of metal ions on its electrophoretic mobility (26). The effects RNAs. The sequence motif, with three duplex stems and a of metal ions on the formation, upon subsequent addition of conserved core of two non-helical segments that are respon- 2+ Mg ions, of an active complex between a hammerhead sible for the self-cleavage reaction (cis-action), was first recog- ribozyme and its substrate can also be monitored by NMR nized in the satellite RNAs of certain viruses (3). Engineered spectroscopy (143–152). trans-acting hammerhead ribozymes, consisting of antisense sections (stem I and stem III) and a catalytic core with a Binding sites for metal ions have been identified by flanking stem–loop II section (Fig. 1A, left), have been used in capturing metal ions within the crystal structure, and such mechanistic studies and tested as potential therapeutic agents capture provides an indication of the importance of the metal 2+ (13,46). Hammerhead ribozymes cleave their target RNAs at ions in catalysis (99,132–136). It was proposed that a Mg ion specific sites, generating a 2′,3′-cyclic phosphate and a 5′-OH binds to the pro-Rpoxygenofthe 5′-phosphate of A (P9 phos- terminus. The NUH rule, where N can be any nucleotide and H phate) with further hydrogen bonding associated with N7 of 2+ can be A, U or C, was originally proposed to define sites of G (153–155). Another site for a Mg ion was localized in 10.1 cleavage, with the most efficient cleavage occurring at GUC the vicinity of the cleavage site. It was proposed that, at this 2+ triplets (125–130). However, the NUH rule was reformulated cleavage site, a Mg ion binds directly to the pro-Rpoxygenof into the NHH rule since other triplets, such as GAC and GCC, the scissile phosphate. Although this possibility remains to be can also be cleaved by a hammerhead ribozyme (131). confirmed, the function of this second metal ion near the Over the past few years, several attempts have been made to scissile phosphate has been proposed to be activation of the determine the overall global structure of hammerhead attacking 2′-OH in the transition state. The site of yet another ribozymes (99,132–137). Although initial structural studies metal ion has also been proposed. Such an ion would act as a indicated that possible configurations of the scissile phosphate switch that induces the conformational changes required to did not allow for an in-line attack mechanism, recent crystallo- achieve the transition state; it would be located adjacent to G graphic studies of a ribozyme with a product or with a modifi- in the catalytic core. This last putative site was identified from 3+ cation (known as a kinetic bottleneck modification) adjacent to an analysis of the kinetics of a Tb inhibition experiment and the cleavage site succeeded in trapping an intermediate that more the elucidation of the crystallographic structure of the complex closely resembled the transition state (137; reviewed in 138). (136). The coordination of a metal ion at this site in solution However, even in this case, the intermediate cannot be consid- was also investigated by lanthanide luminescence spectros- ered as a real transition-state intermediate. In all crystals of copy (156). An additional metal ion binding site in the ribozymes examined to date, a γ-shaped configuration has been hammerhead ribozyme has also been identified by PNMR identified, with stem I forming an acute angle with stem II, and spectroscopy (149). In this case, the metal ion is associated stems II and III being stacked coaxially to form a pseudo-A- with the A phosphate in the catalytic core with an apparent K 13 d form helix, in agreement with results inferred from studies of of 250–570 µ M. However, the exact role of this metal ion fluorescence energy transfer and from electrophoretic and remains unclear. It seems likely that it might be involved in Nucleic Acids Research, 2001, Vol. 29, No. 9 1823 2+ 2+ structural folding since a structural change was detected at this no preference was detected for either Mg or Mn ions site upon the binding of the metal ion. (100,101). However, the 5′-S almost-DNA substrate basically The importance of the binding of a metal ion at the P9 phos- consisted of DNA and the ribozyme reaction with this substrate had an unusually limited dependence on pH. Thus, phate, not only in the ground state but also in the transition we might expect that observed rates of reaction might reflect state, was demonstrated by kinetic analysis with a modified hammerhead ribozyme with a phosphorothioate modification steps other than the chemical cleavage step. In the case of a 5′-S RNA substrate consisting only of RNA, the rate of at this site (157,158) and, in parallel, by analysis with a ribozyme-mediated cleavage of the 5′-S RNA substrate in the ribozyme with an abasic mutation at this site (159,160). The 2+ 2+ binding of a metal ion (Cd )to the Rpsulfurofthe P9 phos- presence of Mg ions was higher by almost two orders of magnitude than that of cleavage of the natural substrate (56). If phorothioate in the transition state was much stronger than the TS1 were a higher energy state than TS2 in the ribozyme reac- binding in the ground state, suggesting the existence of addi- tional ligands for the metal ion in the transition state. More- tion with the natural substrate, the cleavage rate for the 5′-S RNA substrate should be similar to that for the natural substrate over, our own studies indicate strongly that the binding of a because the 5′-bridging phosphorothioate linkage would not be metal ion to N7 of G is catalytically important but not indis- 10.1 pensable: cleavage still occurred with a minimally modified expected to enhance the attack by the 2′-OH (53). By contrast, if TS2 were a higher energy state than TS1 in the ribozyme ribozyme, in which N7 of G was merely replaced by C7 10.1 reaction with the natural substrate, we would expect that the (introduction of an N7-deazaguanine residue to prevent the metal ion from binding to this site) (161). Cleavage was rate of cleavage of the 5′-S RNA substrate would be much higher than that of the natural RNA substrate because a retarded, with ∼30-fold reduction in the rate of cleavage by the mercapto group is a better leaving group than a hydroxyl modified ribozyme. By contrast, a 1000-fold reduction in the cleavage rate resulted from the introduction of an Rp-phos- group. On the basis of these considerations, the results indicate that TS2 is a higher energy state than TS1 in the reactions of phorothioate at the P9 site (157). hammerhead ribozymes with natural substrates, as indicated in It was proposed very recently that the first metal ion that Figure 2. binds to the P9 phosphate in the ground state shifts toward a non-bridging pro-Rp oxygen at the scissile phosphate during Catalytic metal ions in reactions catalyzed by the reaction and binds to this pro-Rp oxygen in the transition hammerhead ribozymes: single-metal-ion mechanisms state. This scenario is consistent with the prediction that additional binding to this P9 metal ion must occur in the tran- In the case of the proteinaceous enzyme RNase A, which does sition state, as mentioned above. Furthermore, it was also not require metal ions as cofactors, the acid/base catalysts are proposed that the metal ion at P9 must be involved directly in provided by two histidine residues within the catalytic pocket the chemical cleavage step, acting as a base catalyst in the tran- (Fig. 5A). Such acid/base functionality can, in principle, be 2+ sition state (27), despite the fact that the P9 metal ion is located replaced by Mg -bound water molecules. The generally at a distance of ∼20 Å from the scissile phosphate in the ground accepted mechanism of hammerhead ribozyme reactions is a state. However, these proposals have been questioned by other single-metal-ion mechanism (27,45,99–103). In this proposed 2+ investigators (162,163). mechanism, the hydroxide ion of a hydrated Mg ion acts as a 2+ 2+ Although it was predicted that a hydrated Mg ion should general base to deprotonate the attacking 2′-OH; the Mg ion participate directly in catalysis, acting as a base catalyst in coordinates directly to the pro-Rp oxygen at the scissile phos- deprotonation of the 2′-OH of C , no crystal structure has been phate, acting as an electrophilic catalyst in TS1; and it is not a obtained with a trapped metal ion located at this cleavage metal ion but a proton that acts as a general acid to stabilize position that might confirm the direct involvement of such a TS2 (Fig. 5B). metal ion in deprotonation of the 2′-OH (137). The single-metal-ion mechanism is supported by the fact that no metal ion was found close to the 5′-leavingoxygeninthe The rate-limiting departure of the 5′-oxygen in reactions original crystallographic structure of a freeze-trapped confor- catalyzed by hammerhead ribozymes mational intermediate of a hammerhead ribozyme (99). Based In the non-enzymatic hydrolysis of a natural RNA, the upon the crystal structure and a molecular dynamic simulation, cleavage of the P-O(5′) bond is the overall rate-limiting step in a different single-metal-ion mechanism was proposed as hydrolysis. By contrast, attack by the 2′-OH on the phosphorus follows (102,103). It was suggested that the involvement of atom is the rate-limiting step with a 5′-S substrate that contains just one metal ion in the transition state might be sufficient for 2+ a phosphorothiolate substitution (54–56,164,165) since, as cleavage. In this model, one Mg ion coordinates simultane- mentioned above, the latter is hydrolyzed much more rapidly ously and directly to the pro-Rpoxygenand to the 2′-attacking than the natural substrate (Fig. 2). The same technique as that oxygen at the cleavage site, acting as a Lewis acid to enhance used to draw these conclusions was used to determine the rate- the deprotonation of the 2′-OH and the subsequent attack by limiting step in reactions catalyzed by hammerhead ribozymes. the nucleophile on the phosphorus atom and/or to stabilize the It was reported that, in the hammerhead-catalyzed cleavage transition state. In addition, it was also proposed that one of the reaction, the departure of the 5′-leaving group is not rate- outer-sphere water molecules that surrounds the metal ion limiting and that a metal cofactor does not interact with the might be located at a position such that it can act as a general leaving group (45,100,101). This conclusion was based on acid to donate a proton to the 5′-leaving group (102,103) experiments with the 5′-S almost-DNA substrate, that was an Another model for cleavage by hammerhead ribozymes was oligodeoxynucleotide substrate that contained a 5′-bridging proposed that was based on the results of molecular dynamic phosphorothiolate linkage adjacent to one ribonucleotide at the studies (166). This model involves two metal ions but it is cleavage site (45). No appreciable thio effect was observed and reminiscent of the single-metal-ion mechanism. In this model, 1824 Nucleic Acids Research, 2001, Vol. 29, No. 9 to the pro-Rp oxygen at the scissile phosphate bond (5,25,162,168,169). Studies of solvent isotope effects and kinetic analysis of a modified substrate (phosphorothiolate; 5′-S substrate), with a 5′-mercapto leaving group at the cleavage site, provided strong support for the double-metal-ion mechanism of catalysis (20,22,25). The overall transition state structure in the hammerhead cleavage reaction is TS2, regardless of whether the reaction proceeds via a concerted one-step mechanism or via a two-step mechanism with a stable pentacoordinated intermediate. Our analysis with a 5′-S substrate demonstrated that it is important that any enzyme that catalyzes the hydrolysis of RNA should stabilize TS2 by donating a proton to the 5′-leaving oxygen or by ensuring coordination of a metal ion to the leaving oxygen. This acid catalysis by a metal ion is also supported by a 2+ recently determined crystallographic structure, in which a Co ion was located close to the 5′-leaving-oxygen atom of the scissile phosphate (134). Figure 6A shows experimentally derived profiles of pH versus rate for reactions in H Oand D O (20,25,169). The 2 2 magnitude of the apparent isotope effect (ratio of rate constants in H Oand D O) is 4.4 and the profiles appear to support the 2 2 2+ possibility that a proton is transferred from (Mg -bound) water molecules. However, careful analysis led us to conclude that a metal ion binds directly to the 5′-oxygen. Since the concentration of the deprotonated 2′-oxygen in H Oshould be higher than that in D O at a fixed pH, we must take into account D2O H2O this difference in pK ,namely, ∆ pK (pK –pK ), when we a a a a analyze the solvent isotope effect of D O (20,25,169,170). We Figure 5. (A) The mechanism of cleavage by ribonuclease A. Two imidazole residues function as general acid–base catalysts. (B) The single-metal-ion can estimate the pK in D O from the pK in H Ousing the a 2 a 2 mechanism proposed for cleavage by the hammerhead ribozyme. One metal linear relationship shown in Figure 6B (6,25,169–172). If the ion binds directly to the pro-Rp oxygen and functions as a general base cata- 2+ pK for a Mg -bound water molecule in H O is 11.4, the ∆ pK a 2 a lyst. (C) The double-metal-ion mechanism proposed for cleavage by the ham- is calculated to be 0.65 (Fig. 6B, red line). Then, the pK in merhead ribozyme. Two metal ions bind directly to the 2′-and 5′-oxygens. a D O should be 12.0. Demonstrating the absence of an intrinsic isotope effect (k /k = 1), the resultant theoretical curves H2O D2O two metal ions are bridged by a hydroxide ion (the µ -hydroxo- closely fit the experimental data, with an apparently ∼4-fold 2+ bridged Mg cluster). One of the metal ions, located near the difference in observed rate constants (Fig. 6A). This result scissile phosphate, binds to the pro-Rp oxygen to act as an indicates that no proton transfer occurs in the hammerhead – 2+ electrophilic catalyst. The bridging OH between the two Mg ribozyme-catalyzed reaction in the transition state and supports 2+ ions abstracts the proton from the Mg -bound water molecule. the hypothesis that the metal ions function as Lewis acids. In 2+ Then the activated hydroxide ion associated with the Mg ion Figure 6A, the apparent plateau of rate constants above pH 8 deprotonates the proximal 2′-OH at the cleavage site to acti- reflects the disruption of the active hammerhead complex by vate the nucleophile, acting as a base catalyst. the deprotonation of uridine and guanosine residues. As described in the previous section, formation of TS2 is the The double-metal-ion mechanism is also supported by rate-limiting step in non-enzymatic reactions (25,54–56). results reported by Pontius et al. (21) and Lott et al. (24). They Thus, TS2 must somehow be stabilized energetically for effective 2+ pointed out the minimal likelihood of base catalysis by Mg - catalysis. Therefore, the hypothesis that reactions catalyzed by bound hydroxide that deprotonates the attacking 2′-OH and the hammerhead ribozymes involve only a general base is insuffi- strong likelihood of Lewis acid catalysis by the direct coordi- cient. 2+ nation of a Mg ion with the attacking 2′-OH, which enhances the deprotonation of the nucleophilic 2′-OH. Their suggestions Catalytic metal ions in reactions catalyzed by were based on the inverse correlation between the rate of hammerhead ribozymes: double-metal-ion mechanisms cleavage and the pK of the added metal ions. Their argument Double-metal-ion mechanisms, in which two metal ions are was as follows. One of the observations used to support the involved in the chemical cleavage step, have been proposed by single-metal-ion mechanism of catalysis (Fig. 5B) is that the numerous groups of investigators (5,18–22,24,25,104). From lower the pK , the higher is the cleavage rate at a given concen- molecular orbital calculations and kinetics analysis, our group tration of metal ions. Although metal ions with lower pK 2+ postulated that the direct coordination of Mg ions with the values might be present at higher concentrations in the form of attacking or the leaving oxygen might promote formation or solvated metal hydroxides at a given pH, such ions should be cleavage of the P-O bond, with these ions acting as Lewis acids correspondingly weaker bases and, therefore, they should be (Fig. 5C) (5,20,22,25,167). Moreover, we excluded the less able to remove the proton from the 2′-OH. As a result, the possible coordination of metal ions, as electrophilic catalysts, effect of concentration would be reduced by the effect of Nucleic Acids Research, 2001, Vol. 29, No. 9 1825 Figure 6. (A) The dependence on pH of the deuterium isotope effect in the hammerhead ribozyme-catalyzed reaction. Green circles show rate constants in H O; yellow circles show rate constants in D O. Solid curves are experimentally determined. The apparent plateau of cleavage rates above pH 8 is due to disruptive 2+ effects on the deprotonation of U and G residues. Dotted lines are theoretical lines calculated from pK values of hydrated Mg ions of 11.4 in H O and 12.0 in a 2 D O and on the assumption that here is no intrinsic isotope effect (α = k /k =1; where α is the coefficient of the intrinsic isotope effect). The following 2 H2O D2O (pKa(base) – pL) (pL – pKa(acid)) equation was used to plot the graph of pL versus log(rate): log k = log (k ) – log[1 + 10 ] – log[1 + 10 ]. In this equation, k is the rate obs max max constant in the case of all acid and base catalysts in active forms: in H O, k = k ; and in D O, k = k = k /α.(B) The isotope effects on the acidities 2 max H2O 2 max D2O H2O D2O H2O 2+ (pK – pK ) of phenols and alcohols as a function of their acid strengths (pK ). The pK of hydrated Mg ions in H O is 11.4, and the red arrow indicates a a a a 2 2+ the isotope effect of 0.65 that results in the pK of hydrated Mg ions in D O being 12.0. The pK of the N3 of cytosine in H O is 6.1 and the blue arrow indicates a 2 a 2 the isotope effect of 0.53 that results in the pK of N3 of cytosine in D O being 6.6. a 2 basicity. In other words, the dependence on pK cannot be ion, coordinates not only with the 5′-leaving oxygen but also, adequately explained by the hypothesis that the solvated metal directly, with the 2′-attacking oxygen [(c) in Fig. 7A]. 3+ hydroxide acts as a base in catalysis. By contrast, deprotona- Although the La ion, which is more positively charged than 2+ tion of the 2′-OH can be greatly accelerated by its direct the Mg ion, might enhance the deprotonation of 2′-OH and, binding to a metal ion, in particular, a metal ion with a rela- as a result, might increase the equilibrium concentration of 2′-O , tively low pK , because the pK of a 2′-OH with a bound metal it would also decrease the nucleophilicity of 2′-O toward the a a ion can be reduced by several units. Such arguments are further electropositive phosphorus. Because the coordination of a supported by ab initio molecular orbital calculations (173). trivalent metal ion at this position reduces the nucleophilicity of the resulting 2′-O much more dramatically than would be Further evidence for a double-metal-ion mechanism was expected for a series of divalent ions and since this negative based on an analysis of the effects of changes in the concentra- 3+ effect is greater than that of an induced higher concentration of tion of La ions in the presence of a fixed concentration of – 3+ 2+ 3+ 2′-O , a higher concentration of La ions decreases the overall Mg ions (24). Analysis of the effects of added La ions rate of cleavage [(c) in Fig. 7A]. yielded a bell-shaped curve, with activation and then inhibition of cleavage (Fig. 7A). Under the conditions of the experiment, However, it has been demonstrated that the binding of a 3+ 2+ La and Mg ions competed for coordination to restricted metal ion to the pro-Rp oxygen of the phosphate moiety of sites that are catalytically important for cleavage by the nucleotide A andtoN7 ofnucleotide G is critical for effi- 9 10.1 3+ 3+ ribozyme. At lower concentrations of La ,aLa ion rather cient catalysis, despite the considerable distance (∼20 Å) 2+ than a Mg ion coordinates to the 5′-leaving oxygen and between the P9 phosphate and the scissile phosphate in the absorbs the negative charge that accumulates at that position in ground state (27,99,128,132–134,157–160). In earlier discus- 3+ 3+ the transition state [(b) in Fig. 7A]. The fully hydrated La ion sions of the above-mentioned La -titration issue (24), it was 2+ 3+ has a pK of 9, which is >2 lower than that of Mg .Whenthe difficult to completely exclude the possibility that La ions 3+ leaving oxygen is directly coordinated with a trivalent La ion, might replace the metal ion at the P9 site and, as a result, might it is a better leaving group than when coordinated with a create the conditions represented by the bell-shaped curve 2+ divalent Mg ion. This difference results in a decline in the shown in Figure 7A. In order to clarify this situation, we examined relative energy of TS2 and acceleration of cleavage. However, a chemically synthesized hammerhead ribozyme (7-deaza-R34) 3+ 3+ 2+ at higher concentrations of La ,aLa ion, instead of a Mg that included a minimal modification, namely, an N7-deaza- 1826 Nucleic Acids Research, 2001, Vol. 29, No. 9 obviously, completely free from potential artifacts due to a 2+ Mg ion coordinated at G . Thus, our results strongly 10.1 support the proposal that a double-metal-ion mechanism is operative in the cleavage reaction that is catalyzed by hammer- head ribozymes (161). Catalytic metal ions as electrophilic catalysts The involvement of a metal ion in a strong interaction with the non-bridging pro-Rp oxygen of the scissile phosphate is generally accepted and has been supported by results from many groups (27,94–97,99,102,103,158,174–176), even though such involvement remains a matter of controversy in light of the work from our laboratory (5,6,25,104,162,168,177). It has been proposed that this metal ion acts as an electrophilic catalyst in the reaction catalyzed by hammerhead ribozymes (Fig. 3e) 2+ on the basis of results of a rescue experiment with Mn ions (94–97), with further support from molecular dynamic studies (102,103,174) and spectroscopic analysis (175). Replacement of the pro-Rp oxygen with sulfur at the cleavage site of a substrate molecule (to yield the RpS substrate for a hammerhead ribozyme) resulted in a large thio effect that 2+ 2+ was relieved by replacement of Mg ions with Mn ions, 2+ which have higher affinity than Mg ions for sulfur. This 2+ observation led to the general conclusion that a Mg ion is directly coordinated with the pro-Rp oxygen. In this arrange- ment, the bound metal ion can act as an electrophilic catalyst and, therefore, the proposed mechanism is very attractive as an explanation for the catalytic activity of metalloenzymes. However, as we have suggested previously, observations of the 3+ thio effect and manganese rescue do not, by themselves, prove Figure 7. Titration with Ln ions. The hammerhead ribozyme reaction was 2+ examined on a background of Mg ions. (A) Data obtained by Lott et al. (24). that the direct coordination of a metal ion with the pro-Rp The proposed binding of metal ions is illustrated. (B) Data obtained by Naka- oxygen at the cleavage site occurs in hammerhead ribozyme- matsu et al. (161). An unmodified ribozyme (R34; red curve) and a modified catalyzed reactions (5,25,168,177). Moreover, although results ribozyme (7-deaza-R34; blue curve) were used. The rate constants were nor- of a reinvestigation of the thio effect using another thiophilic 3+ malized by reference to the maximum rate constant ([La ]=3 µ M). Reactions 2+ metal ion, namely the Cd ion, were used to support the were performed under single-turnover conditions in the presence of 80 nM ribozyme and 40 nM substrate at 37°C. proposed direct coordination of the metal ion with the pro-Rp oxygen of the scissile phosphate (27,158,176), we did not find the argument totally convincing. guanine residue in place of G (Fig. 7B, blue curve) (161). 10.1 In studies that we designed as part of an attempt to explain We compared the kinetic properties of this ribozyme with the thio effect and cadmium rescue by mechanisms that do not those of the parental ribozyme (R34 in Fig. 7B, red curve). involve the direct coordination of a metal ion at the pro-Rp Kinetic analysis revealed that replacement of N7 by C7 at G 10.1 oxygen (162), we found that the rate of ribozyme-catalyzed reduced the catalytic activity but only to a limited extent cleavage of the RpS substrate was ∼1000-fold lower than that (∼30-fold). The most important result, however, was that 2+ of the natural substrate in the absence of thiophilic Cd ions 7-deaza-R34 also yielded a bell-shaped curve upon addition of 2+ and that the addition of the soft Cd ions to the reaction on a 3+ La ions to the reaction mixture. Moreover, the apparent K 2+ 2+ background of hard Mg or Ca ions restored the efficient for the replacement was the same for 7-deaza-R34 and for the cleavage of the RpS substrate (Fig. 8A). Furthermore, at high parental ribozyme R34 (Fig. 7B). These results indicated that 2+ 2+ concentrations of Cd ions on a background of Ca ions, the 2+ binding of a Mg iontoN7ofG was catalytically important 10.1 rate of cleavage of the RpS substrate was similar to that of the but not indispensable for hammerhead ribozyme-mediated natural substrate. 3+ catalysis and, furthermore, they confirmed that the La titra- Although such results might appear to prove the direct coor- tion method, as reported by von Hippel’s group, does indeed dination of a metal ion with the pro-Rp oxygen, we reached a allow monitoring of the actions of catalytic metal ions at the 2+ completely different conclusion. It is true that Cd ions at cleavage site that are directly involved in catalysis. While such 2+ higher concentrations replaced the background Ca ions, and results do not, by themselves, completely exclude the possi- 2+ that, above the apparent dissociation constant of the Cd ions bility that other hypothetical metal-binding sites might have an (K ), the RpS substrate was cleaved at a fixed rate because dapp 2+ 2+ allosteric effect on catalytic activity (136,156,174), the data the Ca ion(s) had been fully replaced by a catalytic Cd 2+ do, at least, increase the likelihood that catalysis by hammer- ion(s). However, it should be noted that Cd ions at higher 2+ head ribozymes involves a double-metal-ion mechanism since concentrations similarly replaced the background Ca ions in 2+ the involvement of a Mg ion at N7 of nucleotide G can be the reaction with the natural substrate and, moreover, that the 10.1 ignored. The data from our experiments with 7-deaza-R34 are, natural substrate (PO) was cleaved at a fixed rate that was identical Nucleic Acids Research, 2001, Vol. 29, No. 9 1827 known to interact with a sulfur atom with a significantly higher affinity (two orders of magnitude higher) than with a hard oxygen atom (162). Estimates can be made of the respective kinetic and thermodynamic parameters in the thermodynamic 2+ cycle (Fig. 8B), as follows. Since the pre-existing Ca ion(s) 2+ within the ribozyme–substrate complex was replaced by a Cd ion(s) with the identical K for both the natural and RpS dapp 2+ substrates, the affinity of the binding of the added Cd ion(s) to each respective complex in the ground state must be the same [K = K ]. Moreover, the cleavage rate in the d GS(PO) d GS(PS) 2+ presence of saturating Cd ions was the same for both the –1 natural and RpS substrates [k = k =1min ]and cleave(PO) cleave(PS) the rate of the non-enzymatic cleavage of the P-O bond in phosphate and phosphorothioate moieties is known to be similar [k = k ; see 178,179]. Because the thermodynamic (PO) (PS) box must be closed (Fig. 8B) and since three out of four param- eters, namely, K , k and k, are the same for both the dGS cleave natural and the RpS substrate, it is apparent that the remaining 2+ parameter (the affinity of the Cd ion for the transition state complex) must also be the same for both substrates [K = d TS(PO) K ], irrespective of whether the cleavage site includes a d TS(PS) regular phosphate or a modified Rp-phosphorothioate moiety. In the case of a modified ribozyme in which the pro-Rp oxygen at the P9 phosphate was replaced by sulfur (RpS-P9 2+ ribozyme), the affinity of Cd ions was higher for the RpS-P9 ribozyme than for the natural ribozyme (27,157,162) [K for dapp the RpS-P9 ribozyme (25 µ M) was smaller than K for the dapp 2+ natural ribozyme (220 µ M) in the presence of 10 mM Mg 2+ ions (157)]. Therefore, a Cd ion does indeed interact with the Rp sulfur at the P9 phosphate but, contrary to the conclusion 2+ reached by other investigators, the Cd ion does not interact with the sulfur atom at the Rp position of the scissile phos- phate, neither in the ground state [K = K ]nor in the 2+ Figure 8. (A) Titration with Cd ions. The hammerhead ribozyme reaction d GS(PO) d GS(PS) 2+ transition state [K = K ]. Thus, it is appropriate to was examined on a background of Ca ions. The substrate had a normal phos- d TS(PO) d TS(PS) phate (natural substrate; blue) or an Rp-phosphorothioate group (RpS sub- emphasize, yet again, that observations of the thio effect and strate; red) at the cleavage site. Solid curves indicate the rate constants for cadmium rescue by themselves are not sufficient to prove that 2+ Cd -associated reactions. Dotted curves indicate the observed rate constants. the direct coordination of a metal ion with the pro-Rpoxygen (B) Thermodynamic boxes for the cleavage of the natural substrate (blue) and at the cleavage site occurs in hammerhead ribozyme-catalyzed the RpS substrate (red) by the hammerhead ribozyme. R, ribozyme–substrate complex with all metal-binding sites occupied except the exchange site(s) reactions. examined here; M, the metal ion(s) that binds to the exchange site(s) examined Numerous experimental results have indicated that the here; K and K , the intrinsic dissociation constants for binding of metal dGS dTS substitution of only one sulfur atom for an oxygen atom has a ion(s) to the exchange site(s) examined here in the ground state and in the tran- major steric effect and that, in many cases, such substitution sition state, respectively; k , the rate of ribozyme-catalyzed cleavage with cleave 2+ Cd ion(s) at the exchange site(s) examined here; and k, the rate of non-enzy- changes the mode of reaction (64,72,88,89,92,149,179–184). matic cleavage. The observed rate constants can be described in terms of With a 3′-S or SpS substrate, the site of cleavage by the RNase pseudo equilibrium constant K s for the formation of the transition state (‡) P ribozyme shifted in the 5′-direction from the modified site from the ground state (k =[k T/h]K , in which k is Boltzmann’s constant, T B d B (the expected cleavage site) to the adjacent unmodified phos- is absolute temperature and h is Planck’s constant). phodiester linkage (88,92), as discussed above. With an SpS substrate, the cleavage site in the group II intron-catalyzed to the rate of cleavage of the RpS substrate (PS) above the K SER reaction also shifted in the 5′-direction (64). A hybridized dapp 2+ 2+ orbital of oxygen or sulfur consists of 2s,2p atomic orbitals or for Cd ions [k = k ]. Clearly, Cd ions enhanced cleave(PO) cleave(PS) 3s,3p atomic orbitals, respectively. The third periodic orbital is the rate of cleavage not only of the RpS substrate but also of the 2+ known to be much bigger than the second one. This difference natural substrate, an indication that Cd ions replaced bound in bulkiness might be responsible for the prevention of 2+ Ca ions even in the case of the natural substrate. Furthermore, the correct positioning of the scissile bond at the active 2+ the K for Cd ions during cleavage of the RpS substrate dapp site. Furthermore, in the case of hammerhead ribozymes, it is was the same as that during cleavage of the natural substrate possible that disruption of the symmetry of the non-bridging [K = K , where GS is the ground state]. d GS(PO) d GS(PS) oxygen by the thio substitution might itself affect the structure 2+ Why was the affinity for Cd ions the same for both the RpS and activity of the ribozyme (179). Introduction of a sulfur and the natural substrate? We would expect the RpS substrate to atom can also result in a significant change in the structure of 2+ have a higher affinity for Cd ions, if the metal ion were directly the complex between the hammerhead ribozyme and its 2+ coordinated with the pro-Rp oxygen, because the soft Cd ion is substrate (150). 1828 Nucleic Acids Research, 2001, Vol. 29, No. 9 2+ 2+ Rescue experiments with Mn or Cd ions do not provide general acid catalysis, as shown by the blue curve in Figure 9A evidence that unequivocally supports electrophilic catalysis in (without a metal hydroxide acting as a general base). This type 2+ hammerhead ribozyme-catalyzed reactions. Thus, Mg ions of profile clearly demonstrates that the observed pK of 6.1 for that catalyze the nucleophilic attack by the 2′-oxygen or that self-cleavage in the presence of divalent metal ions (Fig. 9B, stabilize the 5′-leaving oxygen have no kinetically detectable green curve) reflects the pK of a general acid rather than that 3+ function as simultaneous electrophilic catalysts in hammer- of a general base. It is noteworthy that [Co(NH ) ] inhibited 3 6 2+ head ribozyme-catalyzed reactions, as shown in Figure 5C. the Mg -catalyzed reaction in a competitive fashion, a result 3+ that suggests that [Co(NH ) ] might bind to the same site as 3 6 2+ the functional Mg ion with outer-sphere coordination (note HDV RIBOZYME-CATALYZED REACTIONS 3+ that [Co(NH ) ] does not ionize to yield base catalyst [B:]) 3 6 (42). The similar rate constants determined in the presence of Studies by three groups have revolutionized our understanding 2+ 2+ Ca ions and of Mg ions are also consistent with the action of the mechanism of HDV ribozyme-catalyzed reactions (40–42). of a hydrated metal ion as a Brönsted base rather than as a For the cleavage of phosphodiester bonds, the nucleophile Lewis acid in the reaction catalyzed by the HDV ribozyme must be deprotonated and the leaving group must be proto- (21,42). nated or stabilized by a functional group(s). As illustrated in Figure 4, the developing negative charges in the transition state There was a significant, apparent D O solvent isotope effect in the group I and group II intron-catalyzed reactions are stabi- over the entire range of pH values, confirming that transfer of lized by direct interactions with metal ions. A similar mecha- a proton occurs in the transition state [the ratio of the observed nism is possible for hammerhead ribozyme-catalyzed rate constants, k /k , being as high as 10 (42)]. obs(H2O) obs(D2O) reactions, as shown in Figure 5C. The novel finding with Moreover, since the observed pK for self-cleavage corre- respect to the mechanism of catalysis by the genomic HDV sponded to that for the general acid and since the overall rate- ribozyme is that the pK of cytosine 75 (C ) is perturbed to limiting step appeared to be the cleavage of the bond between a 75 neutrality in the ribozyme–substrate complex and, more the phosphorus and the 5′-leaving oxygen (Fig. 2), the trans- importantly, that C acts as a general acid catalyst in combina- ferred proton in the transition state must have been derived tion with a metal hydroxide which acts as a general base cata- from C . Under the conditions of the measurements, the pK 75 a lyst (Fig. 9). Discovery of this phenomenon provided the first of C in H O was 6.1 and, thus, the estimated ∆ pK was 0.53, 75 2 a direct demonstration that a nucleobase can act as an acid–base as indicated by the blue arrow in Figure 6B. Indeed, in the pL catalyst in an RNA. As a result, as shown by the green curve in profile (pL = pH or pD) in Figure 9B, the pK in D Oisshifted a 2 Figure 9B, the curve that represents the dependence on pH of upward by ∼0.4 ± 0.1 pH units, consistent with the estimated the self-cleavage of the precursor genomic HDV ribozyme has value (42). If we take the pK of C as 6.1inH Oand 6.5 in a 75 2 2+ a slope of unity at pH values below 7 (the activity increases D O and the respective pK values for Mg -bound water to be 2 a linearly as the pH increases, with a slope of +1). Then, at 11.4 and 12.0; and if we assume that the value of the intrinsic higher pH values, the observed rate constant becomes insensi- D O solvent isotope effect is 2 (k /k = 2), we can generate 2 H2O D2O tive to pH. theoretical curves for HDV-catalyzed reactions in H O (green curve) and D O (yellow curve), as shown in Figure 9B. The The slope of unity below pH 7 is consistent with an increase, good agreement of the theoretical curves with the experimen- with pH, in the relative level of the metal hydroxide [B:], tally determined curves (indicated by solid lines in Fig. 9B) which acts as the general base upon deprotonation, and a (42) supports a scenario wherein transfer of a proton does constant amount of the functional protonated form of C [AH], indeed occur from protonated C in the P-O(5′) bond-cleaving which acts as the general acid. The slope of zero from pH 7 to transition state (TS2; Fig. 9C). This conclusion is different pH 9 indicates that the relative level of the metal hydroxide from the conclusion in the case of hammerhead ribozymes [B:] increases (Fig. 9A, red curve), while the relative level of because, in the case of hammerhead ribozymes, the observed, protonated C [AH] decreases by the same amount (Fig. 9A, apparent isotope effect (Fig. 6A) can be fully explained by the blue curve) (42). In agreement with this interpretation, when difference in relative levels of the active species in H Oand in C was replaced by uracil, the resultant C75U mutant, which 75 2 D O without invoking any intrinsic D O solvent isotope effect was unable to assist in the transfer of a proton, did indeed lack 2 2 (k /k =1;Fig.6A). catalytic activity. However, the activity of the C75U mutant H2O D2O 2+ was restored by the addition of imidazole, whose protonated As described above, the HDV ribozyme requires a Mg ion form, the imidazolium ion, is known to act as a good proton for efficient catalysis. The possibility of the coordination of the 2+ donor (41,42). Another mutant, C75A, in which the ring Mg ion with the non-bridging oxygen during the ribozyme- nitrogen N1 at A has a slightly lower pK than the corre- mediated cleavage was examined with RpS and SpS substrates 75 a sponding ring nitrogen N3 of C , supported self-cleavage (185). Determination of the rates of the cleavage steps revealed activity, albeit with reduced efficiency. The observed pK of that the cleavage rates for both substrates were almost the same the C75A mutant was slightly lower than that of the wild-type as that for the natural substrate irrespective of the presence of 2+ 2+ C ribozyme, supporting the interpretation in Figure 9A. Mg ions or Mn ions. These results indicate the absence of a Further convincing evidence for this model comes from the thio effect and the possibility for manganese rescue in the observation that, in the absence of divalent metal ions (in the HDV ribozyme-catalyzed reaction. The association constants 2+ absence of base [B:], see Fig. 9A) and in the presence of a high for Mg ions and the ribozyme–substrate complex were also concentration of monovalent cations (1 M NaCl, 1 mM almost the same for the two substrates. This result supports the 2+ EDTA), the logarithm of the observed rate constant decreased hypothesis that no direct coordination of the Mg ion with any with pH with a slope of –1, as shown by the blue curve in non-bridging oxygen atoms occurs at the scissile phosphate Figure 9B. This observation is consistent with exclusively during cleavage in HDV ribozyme-catalyzed reactions (185), Nucleic Acids Research, 2001, Vol. 29, No. 9 1829 Figure 9. Reactions catalyzed by the genomic HDV ribozyme. (A) Fractions of the active species [AH] that acts as an acid catalyst (blue) and the active species [B:] that acts as a base catalyst (red), respectively. The pK of the acid catalyst is 6.1 and that of the base catalyst is 11.4 in H O. The theoretical curve for H O in (B) was produced a 2 2 by the multiplication of these two curves. (B) Dependence on pH of the deuterium isotope effect in the HDV ribozyme-catalyzed reaction. Green circles, rate constants in H O; yellow circles, rate constants in D O; solid curves, experimental data; dotted curves, theoretical data calculated using the equation in Figure 6 and pK values for C 2 2 a 75 2+ and for hydrated Mg ions of 6.1 and 11.4 in H O and 6.5 and 12.0 in D O, respectively, assuming α = 2. The blue curve is a pH profile in 1 M NaC1 and 1 mM EDTA in 2 2 the absence of divalent metal ions. (C) Energy diagram for cleavage of its substrate by an HDV ribozyme. The rate-limiting step in the reaction with the natural substrate is the cleavage of the P-(5′-O) bond. The structures of transition states TS1 and TS2 are also shown. P(V), the pentacoordinate intermediate/transition state. 1830 Nucleic Acids Research, 2001, Vol. 29, No. 9 2+ and that base catalysis involves the Mg ion with outer-sphere For efficient cleavage, the hairpin ribozyme seems to require not coordination (42). only activation of an attacking nucleophile but also stabilization of the leaving group since the cleavage mechanism is the All the available data support the reaction mechanism for ‘5′-leaving type’, as are the cleavage mechanisms of the HDV HDV ribozyme-catalyzed reactions that is shown in Figure 9C. and hammerhead ribozymes, which involve stabilization of In this model, the donation of a proton is favored and the model TS2. Hairpin ribozymes probably exploit a nucleobase(s) involvesanacidwithanoptimal pK of 7 under physiological instead of divalent metal ions for stabilization of TS2. conditions (42). By contrast, the self-cleavage of the HDV ribozyme in the absence of divalent cations, but in the presence of high concentrations of monovalent cations, suggests that REACTIONS CATALYZED BY RIBOZYMES THAT DO monovalent cations at high concentrations can replace divalent NOTINVOLVE DIVALENT METAL IONS cations in the tertiary folding of the RNA. Thus, divalent Many ribozymes are considered to be metalloenzymes, cations are not absolutely essential for the folding or cleavage 2+ requiring divalent metal ions for efficient catalysis. However, activity of the HDV ribozyme even though a functional Mg cleavage by hairpin ribozymes occurs in the absence of divalent ion does participate in cleavage under physiological condi- metal ions and some small ribozymes can catalyze cleavage in tions. These features appear to be unique among the mecha- the absence of divalent metal ions but in the presence of mono- nisms of action of known ribozymes. It should be emphasized + + + valent cations, such as Na ,Li and NH .Such ribozymes that, since the overall rate-limiting step is the cleavage of the have been called non-obligate metalloenzymes (43,44). It has P-O(5′) bond (TS2), the acid catalysis provided by protonated been suggested that a dense positive charge rather than divalent C is mandatory in reactions catalyzed by the HDV ribozyme metal ions is the general and fundamental requirement for (Fig. 9C). catalysis by a ribozyme (43). Nucleobases are new candidates for acid/base catalysts, as described above for the HDV THE HAIRPIN RIBOZYME ribozyme. In the case of hammerhead ribozymes, typically categorized as metalloenzymes, it seems that reactions can also 2+ The roles of Mg ions in catalysis have been discussed for be catalyzed by monovalent cations but the concentrations of naturally existing ribozymes, such as group I and II introns, such cations have to be very much higher than that of divalent RNase P, HDV and hammerhead ribozymes. The hairpin metal ions if cleavage is to occur at a similar rate. Monovalent ribozyme is exceptional in that the reaction catalyzed by the cations might stabilize the transition state, in particular the hairpin exploits a metal ion in only a passive role. Strong overall rate-limiting formation of TS2, in the same way as experimental evidence for this statement is provided by the divalent metal ions, by providing a dense positively charged observation that the reaction proceeds efficiently in the presence environment at high concentrations. If this is the case, mono- 3+ of [Co(NH ) ] , polyamines or aminoglycoside antibiotics, 3 6 valent cations would be very ineffective under physiological and in the absence of additional metal ions (34–39). The pK of conditions, at least in the case of reactions catalyzed by 3+ the coordinated NH groups of [Co(NH ) ] is estimated to be 3 3 6 hammerhead ribozymes. >14 (186). Moreover, it is an exchange-inert complex (the Important differences between monovalent cations and divalent –10 –1 ligand exchange rate is 10 s ) (186) and it can replace a fully metal ions include not only differences in positive-charge 2+ 3+ hydrated Mg ion since both [Co(NH ) ] and the fully 3 6 density but also differences in the geometry of hydration of the 2+ hydrated Mg ion have octahedral symmetry and similar radii. 2+ ions. Divalent metal ions, such as Mg ions, have octahedral 3+ Thus, it seems unlikely that [Co(NH ) ] functions as a + 3 6 coordination, while monovalent metal ions, such as Na and general base or a Lewis acid to deprotonate the 2′-OH for + Li ions, likely have tetrahedral coordination on the basis of the 3+ nucleophilic attack and it is likely that [Co(NH ) ] supports 3 6 valence bond theory (186,188). These features of monovalent the structure of the RNA appropriately in the same way as the metal ions might not be suitable geometrically for formation of 2+ hydrated Mg ions do. a catalytically competent structure in the transition state such 2+ Other evidence supporting the putative passive (non-cata- as the one shown in Figure 5C (with Mg ions being replaced + + lytic) role of metal ions is that ribozyme-catalyzed cleavage in by Na or Li ions). Therefore, it is likely that a very high 2+ thepresenceofMg ions exhibits no preference for the RpS concentration of such monovalent cations would be required to versus the SpS substrate (34). This result suggests that non- stabilize the overall transition state TS2. Thus, it is very difficult bridging oxygen atoms at the scissile phosphate do not interact to envision, for example, that a hammerhead ribozyme might with metal ions, at least in any direct manner, during cleavage. use monovalent cations as a catalytic cofactor under physio- Additional evidence for a passive role comes from the observa- logical conditions in vivo. Under physiological conditions, it is tion that the ribozyme reaction can occur in films of partially probable that hammerhead ribozymes are metalloenzymes that hydrated RNA in the absence of divalent cations (39). This require divalent cations for catalysis. result indicates that all elements essential for catalytic function are provided by the folded RNA itself. Taken together, all the CONCLUSIONS results support the hypothesis that a nucleobase(s) functions as a general acid/base catalyst. The profile of pH versus rate for The mechanisms exploited by naturally existing ribozymes hairpin ribozymes has two pK values (5.4 and 9.8), with a flat appear to be more diverse than originally anticipated. It is clear, pH-independent region at neutral pH (35). The importance of however, that at least two effective catalysts are necessary to the exocyclic 2-amino group of G in the substrate has been facilitate the overall pathway for the efficient hydrolysis of reported (187). Efforts are being made to identify a nucleo- RNA. 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(1996) Mechanistic ribozymes. Nat. Biotechnol., 16, 961–965. studies on hammerhead ribozymes. In Eckstein,F. and Lilley,D.M.J. (eds), 148.Scott,W.G. (1998) RNA catalysis. Curr. Opin. Struct. Biol., 8, 720–726. Nucleic Acids and Molecular Biology. Catalytic RNA, Vol. 10. Springer, 149.Hansen,M.R., Simorre,J.P., Hanson,P., Mokler,V., Bellon,L., Beigelman,L. New York, NY, pp. 217–230. and Pardi,A. (1999) Identification and characterization of a novel high affinity 171.Bell,R.P. and Kuhn,A.T. (1963) Dissociation constants of some acids in metal-binding site in the hammerhead ribozyme. RNA, 5, 1099–1104. deuterium oxide. Trans. Faraday Soc., 59, 1789–1793. 150.Suzumura,K., Warashina,M., Yoshinari,K., Tanaka,Y., Kuwabara,T., 172.Jenckes,W.P. (1969) Catalysis in Chemistry and Enzymology.McGraw- Orita,M. and Taira,K. (2000) Significant change in the structure of a Hill, New York, NY, pp. 250–253. ribozyme upon introduction of a phosphorothioate linkage at P9: NMR 173.Lyne,P.D. and Karplus,M. (2000) Determination of the pK of the 2′-hydroxyl reveals a conformational fluctuation in the core region of a hammerhead group of a phosphorylated ribose: implications for the mechanism of ribozyme. FEBS Lett., 473, 106–112. hammerhead ribozyme catalysis. J. Am. Chem. Soc., 122, 166–167. 151.Maderia,M., Hunsicker,L.M. and DeRose,V.J. (2000) Metal-phosphate 174.Grasby,J.A., Jonathan,P., Butler,G. and Gait,M.J. (1993) The synthesis of interactions in the hammerhead ribozyme observed by PNMR and oligoribonucleotides containing O -methylguanosine: the role of phosphorothioate substitutions. Biochemistry, 39, 12113–12120. conserved guanosine residues in hammerhead ribozyme cleavage. Nucleic 152.Tanaka,Y., Morita,E.H., Hayashi,H., Kasai,Y., Tanaka,T. and Taira,K. Acids Res., 21, 4444–4450. (2000) Well-conserved tandem G·A pairs and the flanking C·G pair in 175.Cunningham,L.A., Li,J. and Li,Y. (1998) Spectroscopic evidence for hammerhead ribozymes are sufficient for capture of structurally and inner-sphere coordination of metal ions to the active site of a hammerhead catalytically important metal ions. J. Am. Chem. Soc., 122, 11303–11310. ribozyme. J. Am. Chem. Soc., 120, 4518–4519. 153.Ruffner,D.E. and Uhlenbeck,O.C. (1990) Thiophosphate interference 176.Scott,E.C. and Uhlenbeck,O.C. (1999) A re-investigation of the thio effect experiments locate phosphates important for the hammerhead RNA self- at the hammerhead cleavage site. Nucleic Acids Res., 27, 479–484. cleavage reaction. Nucleic Acids Res., 18, 6025–6029. 177.Zhou,D.M., He,Q.C., Zhou,J.M. and Taira,K. (1998) Explanation by a 154.Ruffner,D.E., Stormo,G.D. and Uhlenbeck,O.C. (1990) Sequence 2+ putative triester-like mechanism for the thio effects and Mn rescues in requirements of the hammerhead RNA self-cleavage reaction. reactions catalyzed by a hammerhead ribozyme. FEBS Lett., 431, 154–160. Biochemistry, 29, 10695–10702. 178.Ora,M., Peltomäki,M., Oivanen,M. and Lönnberg,H. (1998) Metal-ion- 155.Tuschl,T. and Eckstein,F. (1993) Hammerhead ribozymes: importance of promoted cleavage, isomerization and desulfurization of the stem–loop II for activity. Proc. Natl Acad. Sci. USA, 90, 6991–6994. diastereomeric phosphoromonothioate analogues of uridylyl(3′,5′)uridine. 156.Feig,A.L., Panek,M., Horrocks,W.D.,Jr and Uhlenbeck,O.C. (1999) J. Org. Chem., 63, 2939–2947. Probing the binding of Tb(III) and Eu(III) to the hammerhead ribozyme 179.Derrick,W.B., Greef,C.H., Caruthers,M.H. and Uhlenbeck,O.C. (2000) using luminescence spectroscopy. Chem. Biol., 6, 801–810. Hammerhead cleavage of the phosphorodithioate linkage. Biochemistry, 157.Peracchi,A., Beigelman,L., Scott,E.C., Uhlenbeck,O.C. and Herschlag,D. 39, 4947–4954. (1997) Involvement of a specific metal ion in the transition of the 180.Warnecke,J.M., Green,C.J. and Hartmann,R.K. (1997) Role of metal ions hammerhead ribozyme to its catalytic conformation. J. Biol. Chem., 272, in the cleavage mechanism by the E.coli RNase P holoenzyme. Nucl. 26822–26826. Nucl., 16, 721–725. 158.Knöll,R., Bald,R. and Fürste,J.P. (1997) Complete identification of nonbridging 181.Brautigam,C.A. and Steitz,T.A. (1998) Structural principles for the phosphate oxygens involved in hammerhead cleavage. RNA, 3, 132–140. inhibition of the 3′-5′ exonuclease activity of Escherichia coli DNA 159.Peracchi,A., Beigelman,L., Usman,N. and Herschlag,D. (1996) Rescue of polymerase I by phosphorothioates. J. Mol. Biol., 277, 363–377. abasic hammerhead ribozymes by exogenous addition of specific bases. 182.Hamm,M.L., Schwans,J.P. and Piccirilli,J.A. (2000) The hammerhead Proc. Natl Acad. Sci. USA, 93, 11522–11527. ribozyme catalyzes the deglycosylation of 2′-mercaptocytidine. J. Am. 160.Peracchi,A., Karpeisky,A., Maloney,L., Beigelman,L. and Herschlag,D. Chem. Soc., 122, 4223–4224. (1998) A core folding model for catalysis by the hammerhead ribozyme 183.Hamm,M.L., Nikolic,D., van Breemen,R.B. and Piccirilli,J.A. (2000) accounts for its extraordinary sensitivity to abasic mutations. Unconventional origin of metal ion rescue in the hammerhead ribozyme 2+ Biochemistry, 37, 14765–14775. reaction: Mn -assisted redox conversion of 2′-mercaptocytidine to 161.Nakamatsu,Y., Kuwabara,T., Warashina,M., Tanaka,Y., Yoshinari,K. and cytidine. J. Am. Chem. Soc., 122, 12069–12078. Taira,K. (2000) Significant activity of a modified ribozyme with N7- 184.Smith,J.S. and Nikonowicz,E.P. (2000) Phosphorothioate substitution can deazaguanine at G : the double-metal-ion mechanism of catalysis in substantially alter RNA conformation. Biochemistry, 39, 5642–5652. 10.1 reactions catalysed by hammerhead ribozymes. Genes Cells, 5, 603–612. 185.Hamid,F., Kawakami,J., Nishikawa,F. and Nishikawa,S. (1997) Analysis 162.Yoshinari,K. and Taira,K. (2000) A further investigation and reappraisal of the cleavage reaction of a trans-acting human hepatitis delta virus of the thio effect in the cleavage reaction catalyzed by a hammerhead ribozyme. Nucleic Acids Res., 25, 3124–3130. ribozyme. Nucleic Acids Res., 28, 1730–1742. 186.Basolo,F. and Pearson,R.G. (1967) Mechanism of Inorganic Reactions. A Study 163.Murray,J.B. and Scott,W.G. (2000) Does a single metal ion bridge the A- of Metal Complexes in Solution. John Wiley and Sons, Inc, New York, NY. 9 and scissile phosphate groups in the catalytically active hammerhead 187.Chowrira,B.M., Berzal-Herranz,A. and Burke,J.M. (1991) Novel guanosine ribozyme structure? J. Mol. Biol., 296, 33–41. requirement for catalysis by the hairpin ribozyme. Nature, 354, 320–322. 164.Uchimaru,T., Stec,W.J., Tsuzuki,S., Hirose,T., Tanabe,K. and Taira,K. 188.Matsushima,Y. and Takashima,Y. (1999) Seimei no Mukikagaku. (1996) Ab initio investigation on nucleophilic ring opening of 1,3,2- Hirokawa Publishing Co, Tokyo, Japan. Nucleic Acids Research, 2001, Vol. 29, No. 9 1835 Figure 1. The two-dimensional structures of various ribozymes. The ribozyme or intron portion is printed in green. The substrate or exon portion is printed in black. Arrows indicate sites of cleavage by ribozymes. (A) Left, the two-dimensional structure of a hammerhead ribozyme and its substrate. Outlined letters are conserved bases that are involved in catalysis. Right, the γ-shaped structure of the hammerhead ribozyme–substrate complex. (B–F) The two-dimensional struc- tures of a hairpin ribozyme, the genomic HDV ribozyme, a group I ribozyme from Tetrahymena, a group II ribozyme from Saccharomyces cerevisiae (aiγ 5) and scherichiathe ribozyme of RNase P from E.coli, respectively. Figure 2. The two-step reaction scheme for the hydrolysis of a phosphodiester bond in RNA. First, the 2′-oxygen attacks the phosphorus atom, acting as an internal nucleophile, to generate the pentacoordinated intermediate or transtion state TS1. The 5′-oxygen then departs from the intermediate to complete cleavage at TS2. TS1 can be stabilized by a general base catalyst and TS2 can be stabilized by a general acid catalyst, as illustrated at the summits of the energy diagram.These transition states can also be stabilized by the direct binding of Lewis acids to the 2′-attacking oxygen and the 5′-leaving oxygen. Figure 3. Possible catalytic functions of metal ions in the cleavage of a phosphodiester bond. Metal ions can act as (a) a general acid catalyst, (b) a general base catalyst, (c) a Lewis acid that stabilizes the leaving group, (d) a Lewis acid that enhances the deprotonation of the attacking nucleophile and (e) an electrophilic catalyst that increases the electrophilicity of the phosphorus atom. Figure 4. A schematic representation of splicing reactions and the structures of transition states at each step. (A) The group I intron splicing reaction. (i) In the first step, the 3′-OH of the exogenous conserved G attacks the phosphorus at the 5′ splice site and generates the G-attached intron 3′–exon 2 intermediate and a free 5′ exon 1. In the second step, the 3′-OH of the 5′ exon 1 attacks the phosphorus at the 3′ splice site to produce ligated exons and the excised G-attached intron. (ii) 2+ The proposed chemical mechanism of the first step. The 3′-OH of the exogenous G is a nucleophile and the 3′-OH of the U is a leaving group. One of the Mg –1 ions [site (b)] coordinates with the 3′-OH of the G to activate the attacking group. The second [site (c)] coordinates with the 2′-OH of the G. The third [site (d)] coordinates with the pro-Sp oxygen to stabilize the transition state or the intermediate. The fourth [site (a)] coordinates with the 3′-OH of the U to stabilize the –1 leaving group. The 2′-OH also protonates the 3′-leaving oxygen of the U . It is not known whether or not the metal ion at site (d) is the same as those at the other –1 sites, (a), (b) and (c) (72). IGS represents the internal guide sequence. (B) The group II intron splicing reaction. (i) In the first step, the 2′-OH of an A residue that is conserved in the intron attacks the phosphorus at the 5′ splice site and generates an intron 3′–exon2intermediateand afree5′ exon 1. In the second step, the free 3′-OH of the 5′ exon attacks the phosphorus at the 3′ splice site to produce ligated exons and an excised intron. SER indicates the spliced-exon reopening reaction. (ii) The proposed chemical mechanisms of the first and the second steps. In the first step, the 2′-OH of an intron A residue is the nucleophile and the 3′- 2+ OH of the 5′ splice site terminus is the leaving group. One Mg ion coordinates with the 3′-OH to stabilize the leaving group. Other coordinations and/or interac- tions remain to be clarified. In the second step, the 3′-OH of the C, the 5′ splice site terminus, becomes the nucleophile and the 3′-OH of the U is the leaving group. 2+ One Mg ion coordinates directly with the 2′-OH and the 3′-OH of the U. Other coordinations and/or interactions remain to be clarified. Figure 5. (A) The mechanism of cleavage by ribonuclease A. Two imidazole residues function as general acid–base catalysts. (B) The single-metal-ion mechanism proposed for cleavage by the hammerhead ribozyme. One metal ion binds directly to the pro-Rp oxygen and functions as a general base catalyst. (C) The double- metal-ion mechanism proposed for cleavage by the hammerhead ribozyme. Two metal ions bind directly to the 2′-and 5′-oxygens. Figure 6. (A) The dependence on pH of the deuterium isotope effect in the hammerhead ribozyme-catalyzed reaction. Green circles show rate vconstants in H O; yellow circles show rate constants in D O. Solid curves are experimentally determined. The apparent plateau of cleavage rates above pH 8 is due to disruptive 2+ effects on the deprotonation of U and G residues. Dotted lines are theoretical lines calculated from pK values of hydrated Mg ions of 11.4 in H O and 12.0 in a 2 D O and on the assumption that here is no intrinsic isotope effect (α = k /k =1;where α is the coefficient of the intrinsic isotope effect). The following 2 H2O D2O (pKa(base) – pL) (pL – pKa(acid), equation was used to plot the graph of pL versus log(rate): log k = log (k )–log[1+ 10 ] – log[1 + 10 ]. In this equation, k is the rate obs max max constant in the case of all acid and base catalysts in active forms: in H O, k = k ;and in D O, k = k = k /α.(B) The isotope effects on the acidities 2 max H2O 2 max D2O H2O D2O H2O 2+ (pK – pK ) of phenols and alcohols as a function of their acid strengths (pK ). The pK of hydrated Mg ions in H O is 11.4, and the red arrow indicates a a a a 2 2+ the isotope effect of 0.65 that results in the pK of hydrated Mg ions in D O being 12.0. The pK of the N3 of cytosine in H O is 6.1 and the blue arrow indicates a 2 a 2 the isotope effect of 0.53 that results in the pK of N3 of cytosine in D O being 6.6. a 2 3+ 2+ Figure 7. Titration with Ln ions. The hammerhead ribozyme reaction was examined on a background of Mg ions. (A) Data obtained by Lott et al. (24). The proposed binding of metal ions is illustrated. (B) Data obtained by Nakamatsu et al. (161). An unmodified ribozyme (R34; red curve) and a modified ribozyme (7- 3+ deaza-R34; blue curve) were used. The rate constants were normalized by reference to the maximum rate constant ([La ]= 3 µ M). Reactions were performed under single-turnover conditions in the presence of 80 nM ribozyme and 40 nM substrate at 37°C. 2+ 2+ Figure 8. (A) Titration with Cd ions. The hammerhead ribozyme reaction was examined on a background of Ca ions. The substrate had a normal phosphate 2+ (natural substrate; blue) or an Rp-phosphorothioate group (RpS substrate; red) at the cleavage site. Solid curves indicate the rate constants for Cd -associated reactions. Dotted curves indicate the observed rate constants. (B) Thermodynamic boxes for the cleavage of the natural substrate (blue) and the RpS substrate (red) by the hammerhead ribozyme. R, ribozyme–substrate complex with all metal-binding sites occupied except the exchange site(s) examined here; M, the metal ion(s) that binds to the exchange site(s) examined here; K and K , the intrinsic dissociation constants for binding of metal ion(s) to the exchange site(s) examined dGS dTS 2+ here in the ground state and in the transition state, respectively; k , the rate of ribozyme-catalyzed cleavage with Cd ion(s) at the exchange site(s) examined cleave here; and k, the rate of non-enzymatic cleavage. The observed rate constants can be described in terms of pseudo equilibrium constant K s for the formation of the transition state (‡) from the ground state (k =[k T/h]K ,in which k is Boltzmann’s constant and h is Planck’s constant). B d B Figure 9. Reactions catalyzed by the genomic HDV ribozyme. (A) Fractions of the active species [AH] that acts as an acid catalyst (blue) and the active species [B:] that acts as a base catalyst (red), respectively. The pK of the acid catalyst is 6.1 and that of the base catalyst is 11.4 in H O. The theoretical curve for H Oin a 2 2 (B) was produced by the multiplication of these two curves. (B) Dependence on pH of the deuterium isotope effect in the HDV ribozyme-catalyzed reaction. Green circles, rate constants in H O; yellow circles, rate constants in D O; solid curves, experimental data; dotted curves, theoretical data calculated using the equation 2 2 2+ in Figure 6 and pK values for C and for hydrated Mg ions of 6.1 and 11.4 in H O and 6.5 and 12.0 in D O, respectively, assuming α = 2. The blue curve is a a 75 2 2 pH profile in 1 M NaC1 and 1 mM EDTA in the absence of divalent metal ions. (C) Energy diagram for cleavage of its substrate by an HDV ribozyme. The rate- limiting step in the reaction with the natural substrate is the cleavage of the P-(5′-O) bond. The structures of transition states TS1 and TS2 are also shown. P(V), the pentacoordinate intermediate/transition state.

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SURVEY AND SUMMARY: Recent advances in the elucidation of the mechanisms of action of ribozymes

SURVEY AND SUMMARY: Recent advances in the elucidation of the mechanisms of action of ribozymes

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SURVEY AND SUMMARY: Recent advances in the elucidation of the mechanisms of action of ribozymes

SURVEY AND SUMMARY: Recent advances in the elucidation of the mechanisms of action of ribozymes

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