TY - JOUR
AU - Peričin, Draginja
AB - Abstract This review is a summary of our current knowledge of the structure, function and mechanism of action of the three zinc-containing alcohol dehydrogenases, YADH-1, YADH-2 and YADH-3, in baker's yeast, Saccharomyces cerevisiae. The opening section deals with the substrate specificity of the enzymes, covering the steady-state kinetic data for its most known substrates. In the following sections, the kinetic mechanism for this enzyme is reported, along with the values of all rate constants in the mechanism. The complete primary structures of the three isoenzymes of YADH are given, and the model of the 3D structure of the active site is presented. All known artificial mutations in the primary structure of the YADH are covered in full and described in detail. Further, the chemical mechanism of action for YADH is presented along with the complement of steady-state and ligand-binding data supporting this mechanism. Finally, the bio-organic chemistry of the hydride-transfer reactions catalyzed by the enzyme is covered: this chemistry explains the narrow substrate specificity and the enantioselectivity of the yeast enzyme. Yeast alcohol dehydrogenase, Alcohol dehydrogenase, Saccharomyces cerevisiae 1 Introduction Yeast alcohol dehydrogenase (EC 1.1.1.1) is a member of a large family of zinc-containing alcohol dehydrogenases. The primary structures of 47 members of this family have been determined and aligned, and an evolutionary tree has been constructed, assuming a divergent evolution from a common ancestral gene [1]. In this way, it was possible to identify four divergent groups of alcohol dehydrogenases in this family: vertebrates, plants, eukaryotic microorganisms and prokaryotic bacteria. Baker's yeast (Saccharomyces cerevisiae), a member of the third group, has three isoenzymes of alcohol dehydrogenase: YADH-1, YADH-2, and YADH-3. YADH-1 is the constitutive form that is expressed during anaerobic fermentation [2]. YADH-2 is another cytoplasmic form, which is repressed by glucose [3], and YADH-3 is found in the mitochondria [4]. YADH-1 accounts for the major part of alcohol dehydrogenase activity in growing baker's yeast. The structure, function and mechanism of action of yeast alcohol dehydrogenase have been reviewed three decades ago [5,6]. The purpose of this article is to update the subject and to review novel data on the structure, function and mechanism of action of the isoenzyme YADH-1; this isoenzyme will be abbreviated as YADH throughout the text. The steady-state kinetic constants are presented in the nomenclature of Cleland [7]. 2 Isoenzymes of YADH Yeast alcohol dehydrogenase was one of the first enzymes to be purified and isolated [8]. If the steady-state kinetic properties of the ADH isoenzymes are compared, a large degree of similarity is detected. Table 1 shows the steady-state kinetic constants for the three isoenzymes of YADH, isolated from baker's yeast. 1 Steady-state kinetic constants of yeast ADH isoenzymes with ethanol and acetaldehyde as substrates, at pH 7.3, 30°Ca Constant  Unit  YADH-1  YADH-2  YADH-3  V1  s−1  340  130  450  KA  μM  170  110  240  KB  mM  17  0.81  12  V1/KB  mM−1 s−1  20  160  37.5  V2  s−1  1700  1040  2100  KQ  μM  110  50  70  KP  mM  1.1  0.09  0.44  V2/KP  mM−1 s−1  1540  11 550  4770  Constant  Unit  YADH-1  YADH-2  YADH-3  V1  s−1  340  130  450  KA  μM  170  110  240  KB  mM  17  0.81  12  V1/KB  mM−1 s−1  20  160  37.5  V2  s−1  1700  1040  2100  KQ  μM  110  50  70  KP  mM  1.1  0.09  0.44  V2/KP  mM−1 s−1  1540  11 550  4770  aCalculated from the data of Ganzhorn et al. [9]. View Large 1 Steady-state kinetic constants of yeast ADH isoenzymes with ethanol and acetaldehyde as substrates, at pH 7.3, 30°Ca Constant  Unit  YADH-1  YADH-2  YADH-3  V1  s−1  340  130  450  KA  μM  170  110  240  KB  mM  17  0.81  12  V1/KB  mM−1 s−1  20  160  37.5  V2  s−1  1700  1040  2100  KQ  μM  110  50  70  KP  mM  1.1  0.09  0.44  V2/KP  mM−1 s−1  1540  11 550  4770  Constant  Unit  YADH-1  YADH-2  YADH-3  V1  s−1  340  130  450  KA  μM  170  110  240  KB  mM  17  0.81  12  V1/KB  mM−1 s−1  20  160  37.5  V2  s−1  1700  1040  2100  KQ  μM  110  50  70  KP  mM  1.1  0.09  0.44  V2/KP  mM−1 s−1  1540  11 550  4770  aCalculated from the data of Ganzhorn et al. [9]. View Large It is evident that YADH-1 and YADH-3 have very similar kinetic characteristics, while YADH-2 has a much higher substrate specificity for ethanol (V1/KB) and acetaldehyde (V2/KP), and much lower Michaelis constants with ethanol (KB) and acetaldehyde (KP). Recently, the kinetic characterization of YADH-1 and YADH-2 has been extended by measuring their specificity constants (V1/KB) for a number of long-chain alcohols and diols. It was found that for all alcohols, normalized rates with YADH-2 were about three-fold faster than with YADH-1 [10]. 3 Substrate specificity Yeast alcohol dehydrogenase catalyzes the following reversible redox reaction [5]:   1 At neutral pH, the equilibrium is shifted far to the left (Table 2). 2 Steady-state kinetic constants for the oxidation of various alcohols at neutral pH Constant  Unit  Ethanola  Propan-1-ola  Butan-1-ola  Hexan-1-olb  Decan-1-olb  Propan-2-olc  (S)-(+)-Butan-2-olc  Allyl alcohold  Ethyleneglycold  Trise  V1  s−1  454  67  25  15.4  14.4  7  0.9  546  7.0  0.5  KA  μM  109  150  250  169  200  597  376  520  370  698  KiA  μM  325  235  160  152  190  378  398  730  550  842  KB  mM  21.7  29.2  32  3.2  0.1  117  35  14.6  444  6415  V1/KA  mM−1 s−1  4165  447  100  91  72  11.7  2.4  1058  19.2  0.72  V1/KB  mM−1 s−1  20.9  22.9  0.78  4.8  144  0.06  0.026  37.5  0.016  0.0001  V1KiA/KA  s−1  1354  105  16  13.8  13.7  4.4  0.95  766  10.4  0.60  Keqf  –  0.00019  –  0.00027  –  –  0.146  0.40  –  –  –  Constant  Unit  Ethanola  Propan-1-ola  Butan-1-ola  Hexan-1-olb  Decan-1-olb  Propan-2-olc  (S)-(+)-Butan-2-olc  Allyl alcohold  Ethyleneglycold  Trise  V1  s−1  454  67  25  15.4  14.4  7  0.9  546  7.0  0.5  KA  μM  109  150  250  169  200  597  376  520  370  698  KiA  μM  325  235  160  152  190  378  398  730  550  842  KB  mM  21.7  29.2  32  3.2  0.1  117  35  14.6  444  6415  V1/KA  mM−1 s−1  4165  447  100  91  72  11.7  2.4  1058  19.2  0.72  V1/KB  mM−1 s−1  20.9  22.9  0.78  4.8  144  0.06  0.026  37.5  0.016  0.0001  V1KiA/KA  s−1  1354  105  16  13.8  13.7  4.4  0.95  766  10.4  0.60  Keqf  –  0.00019  –  0.00027  –  –  0.146  0.40  –  –  –  aCalculated from the data of Dickinson and Monger [11], at pH 7.0, 25°C. bCalculated from the data of Schöpp and Aurich [12], at pH 8.0, 25°C. cCalculated from the data of Trivić and Leskovac [13], at pH 7.0, 25°C. dCalculated from the data of Trivić and Leskovac [14], at pH 7.0, 25°C. eCalculated from the data of Chen and Huang [15], at pH 8.2, 25°C. fKeq=V1KiQKP/(V2KiAKB). View Large 2 Steady-state kinetic constants for the oxidation of various alcohols at neutral pH Constant  Unit  Ethanola  Propan-1-ola  Butan-1-ola  Hexan-1-olb  Decan-1-olb  Propan-2-olc  (S)-(+)-Butan-2-olc  Allyl alcohold  Ethyleneglycold  Trise  V1  s−1  454  67  25  15.4  14.4  7  0.9  546  7.0  0.5  KA  μM  109  150  250  169  200  597  376  520  370  698  KiA  μM  325  235  160  152  190  378  398  730  550  842  KB  mM  21.7  29.2  32  3.2  0.1  117  35  14.6  444  6415  V1/KA  mM−1 s−1  4165  447  100  91  72  11.7  2.4  1058  19.2  0.72  V1/KB  mM−1 s−1  20.9  22.9  0.78  4.8  144  0.06  0.026  37.5  0.016  0.0001  V1KiA/KA  s−1  1354  105  16  13.8  13.7  4.4  0.95  766  10.4  0.60  Keqf  –  0.00019  –  0.00027  –  –  0.146  0.40  –  –  –  Constant  Unit  Ethanola  Propan-1-ola  Butan-1-ola  Hexan-1-olb  Decan-1-olb  Propan-2-olc  (S)-(+)-Butan-2-olc  Allyl alcohold  Ethyleneglycold  Trise  V1  s−1  454  67  25  15.4  14.4  7  0.9  546  7.0  0.5  KA  μM  109  150  250  169  200  597  376  520  370  698  KiA  μM  325  235  160  152  190  378  398  730  550  842  KB  mM  21.7  29.2  32  3.2  0.1  117  35  14.6  444  6415  V1/KA  mM−1 s−1  4165  447  100  91  72  11.7  2.4  1058  19.2  0.72  V1/KB  mM−1 s−1  20.9  22.9  0.78  4.8  144  0.06  0.026  37.5  0.016  0.0001  V1KiA/KA  s−1  1354  105  16  13.8  13.7  4.4  0.95  766  10.4  0.60  Keqf  –  0.00019  –  0.00027  –  –  0.146  0.40  –  –  –  aCalculated from the data of Dickinson and Monger [11], at pH 7.0, 25°C. bCalculated from the data of Schöpp and Aurich [12], at pH 8.0, 25°C. cCalculated from the data of Trivić and Leskovac [13], at pH 7.0, 25°C. dCalculated from the data of Trivić and Leskovac [14], at pH 7.0, 25°C. eCalculated from the data of Chen and Huang [15], at pH 8.2, 25°C. fKeq=V1KiQKP/(V2KiAKB). View Large Substrate specificity of YADH is restricted to primary unbranched aliphatic alcohols, and any branching in the side chain diminishes the activity of the enzyme and lowers its efficiency. In addition, the enzyme also shows activity towards secondary alcohols. Table 2 presents the steady-state kinetic constants for various alcoholic substrates and Table 3 shows the steady-state constants for various carbonyl substrates of the yeast enzyme. 3 Steady-state kinetic constants for the reduction of various carbonyl substrates at neutral pH Constant  Unit  Acetaldehydea  Butyraldehydea  Acetoneb  Butan-2-one-b  Chloroacetaldehydec  NDMAd  DACAe  V2  s−1  3850  3450  9  0.7  117  2.1  0.176  KQ  μM  96  97  43  38  270  456  46  KiQ  μM  12.5  7  17.5  15.2  74  119  7.6  KP  mM  0.9  27.5  477  285  4  1.5  0.61  V2/KQ  mM−1 s−1  40100  35570  209  18.4  431  4.5  3.8  V2/KP  mM−1 s−1  4280  125  0.019  0.0025  25.2  1.4  0.29  V2KiQ/KQ  s−1  501  249  3.66  0.38  31.9  0.54  0.03  Constant  Unit  Acetaldehydea  Butyraldehydea  Acetoneb  Butan-2-one-b  Chloroacetaldehydec  NDMAd  DACAe  V2  s−1  3850  3450  9  0.7  117  2.1  0.176  KQ  μM  96  97  43  38  270  456  46  KiQ  μM  12.5  7  17.5  15.2  74  119  7.6  KP  mM  0.9  27.5  477  285  4  1.5  0.61  V2/KQ  mM−1 s−1  40100  35570  209  18.4  431  4.5  3.8  V2/KP  mM−1 s−1  4280  125  0.019  0.0025  25.2  1.4  0.29  V2KiQ/KQ  s−1  501  249  3.66  0.38  31.9  0.54  0.03  aCalculated from the data of Dickinson and Monger [11], at pH 7.0, 25°C. bCalculated from the data of Trivić and Leskovac [13], at pH 7.0, 25°C. cCalculated from the data of Leskovac et al. [16], at pH 9.0, 25°C. dCalculated from the data of Trivić et al. [17], at pH 8.9, 25°C. eCalculated from the data of Leskovac et al. [16], at pH 7.0, 25°C. View Large 3 Steady-state kinetic constants for the reduction of various carbonyl substrates at neutral pH Constant  Unit  Acetaldehydea  Butyraldehydea  Acetoneb  Butan-2-one-b  Chloroacetaldehydec  NDMAd  DACAe  V2  s−1  3850  3450  9  0.7  117  2.1  0.176  KQ  μM  96  97  43  38  270  456  46  KiQ  μM  12.5  7  17.5  15.2  74  119  7.6  KP  mM  0.9  27.5  477  285  4  1.5  0.61  V2/KQ  mM−1 s−1  40100  35570  209  18.4  431  4.5  3.8  V2/KP  mM−1 s−1  4280  125  0.019  0.0025  25.2  1.4  0.29  V2KiQ/KQ  s−1  501  249  3.66  0.38  31.9  0.54  0.03  Constant  Unit  Acetaldehydea  Butyraldehydea  Acetoneb  Butan-2-one-b  Chloroacetaldehydec  NDMAd  DACAe  V2  s−1  3850  3450  9  0.7  117  2.1  0.176  KQ  μM  96  97  43  38  270  456  46  KiQ  μM  12.5  7  17.5  15.2  74  119  7.6  KP  mM  0.9  27.5  477  285  4  1.5  0.61  V2/KQ  mM−1 s−1  40100  35570  209  18.4  431  4.5  3.8  V2/KP  mM−1 s−1  4280  125  0.019  0.0025  25.2  1.4  0.29  V2KiQ/KQ  s−1  501  249  3.66  0.38  31.9  0.54  0.03  aCalculated from the data of Dickinson and Monger [11], at pH 7.0, 25°C. bCalculated from the data of Trivić and Leskovac [13], at pH 7.0, 25°C. cCalculated from the data of Leskovac et al. [16], at pH 9.0, 25°C. dCalculated from the data of Trivić et al. [17], at pH 8.9, 25°C. eCalculated from the data of Leskovac et al. [16], at pH 7.0, 25°C. View Large Ethanol is by far the best substrate of the yeast enzyme. Methanol is a very poor substrate of YADH; the methanol activity of the enzyme at pH 8.8 is only 0.07% of its ethanol activity under identical conditions. The enzyme is able to oxidize methanol by NAD+ to formaldehyde and NADH, but the enzymatic reaction is very complex due to interference of numerous side reactions [18]. Allyl and cinnamyl alcohol are, however, excellent substrates; kinetic constants for the latter alcohol are: V1=133 s−1 and V1/KB=29 mM−1 s−1, at pH 8.2, 25°C [19]. (S)-(+)-Butan-2-ol is a much better substrate than (R)-(−)-butan-2-ol (V1=1.0 and 0.05 s−1, and V1/KB=18 and 0.8 M−1 s−1, respectively, at pH 7.3, 30°C) [20]. 4-Methyl-1-pentanol (V1=7 s−1, pH 8.2) is a much better substrate than 2-methyl-1-propanol (V1=0.2 s−1, pH 7.3) or 3-methyl-1-butanol (V1=0.3 s−1, pH 8.2) [19,20]. It was reported that glycerol, glyceraldehyde and acetol are poor substrates of YADH [21], whereas benzyl alcohol and benzaldehyde are extremely poor substrates of this enzyme [20,22]. It has also been reported that p-chlorobenzyl alcohol and p-methoxybenzyl alcohol are slowly oxidized by NAD+ in the presence of YADH [23]. 2-Chloroethanol, 2-fluoroethanol, 2,2,2-trifluoroethanol, propargyl alcohol, glycidol and polyethylene glycol are no substrates of the yeast enzyme [24]. Yeast alcohol dehydrogenase catalyzes three essentially irreversible chemical reactions:   2  3  4 Chloroacetaldehyde is an excellent substrate of YADH (Table 3), while 2-chloroethanol is not oxidized by NAD+, which makes the reaction 2 essentially irreversible [16]. p-Nitroso-N,N-dimethylaniline (NDMA) is readily reduced by NADH, in the presence of YADH (reaction 4); the primary product of this reaction, the corresponding hydroxylamine, is transformed into a quinonediimine compound by the loss of a molecule of water. The last compound is reduced non-enzymatically by NADH to p-amino-N,N-dimethylaniline [17,25]. YADH has a weak aldehyde dehydrogenase activity; it is able to catalyze an irreversible oxidation of acetaldehyde to acetic acid with NAD+, with an apparent kcat=2.3 s−1 and V/K=34 M−1 s−1, at pH 8.8, 22°C [26]. Free acetaldehyde is a true substrate for alcohol dehydrogenase [27], and gem-diol is probably a true substrate for aldehyde dehydrogenase activity of YADH [26]. 4 Steady-state kinetic mechanism Yeast alcohol dehydrogenase catalyzes the chemical reactions described by Eq. 1. Numerous investigations of the steady-state kinetic mechanism of the yeast enzyme have been conducted by several authors [9,11,28–36]; they have led to the conclusion that the yeast enzyme follows the steady-state random mechanism on the alcohol side, and a steady-state ordered mechanism on the aldehyde side of the catalytic cycle, with primary aliphatic alcohols and aldehydes (Scheme 1). Scheme 1 View largeDownload slide Scheme 1 View largeDownload slide The mechanism in Scheme 1 is restricted to primary unbranched aliphatic alcohols and aldehydes, if the latter are present in lower concentrations [33]. The initial rate equation for this mechanism, in the forward direction and in the absence of products, is given by [37]:   5 where X=1+k10/k5. The applicability of Eq. 5 to alcohol oxidation is readily apparent. Eq. 5 predicts that the monomolecular kinetic constant V1 and the bimolecular specificity constants V1/KA and V1/KB are dependent on the nature of substrate B; inspection of data in Table 2 shows that this is true for all primary unbranched alcohols. Also, Eq. 5 predicts that the inhibitory constant KiA is dependent on the nature of substrate B and, therefore, cannot be equal to the dissociation constant of the E·NAD+-complex; Table 2 shows that this is true for all the above alcohols. In addition, a direct determination of KE,NAD+ shows that it is not equal to KiA-, in any case (Fig. 4). The initial-rate equation in the reverse direction, reduction of aldehydes, and in the absence of substrates of reaction, is given by the general expression for the steady-state ordered mechanism [38]:   6 where Y=1+k9/k4. 4 View largeDownload slide The Brändén mechanism. 4 View largeDownload slide The Brändén mechanism. Eq. 6 satisfies the results obtained for the reduction of acetaldehyde and butyraldehyde in predicting a linear reciprocal equation, in which the KiQ, V2/KQ and V2KiQ/KQ constants are independent of the nature of the aldehyde (Table 3). The kinetic mechanism in Scheme 1 is compatible with deuterium isotope effects on maximal rates reported for ethanol, DV1=1.8, DV1/KA=1.8, and DV1/KB=3.2 [39], propan-1-ol, DV1=3.7 [40], butan-1-ol, DV1=3.7 [41], and propan-2-ol, DV1=2.2 around neutrality [13]. With ethanol, the effect on DV1/KA was smaller than on DV1/KB, suggesting that NAD+ binds before ethanol; the still significant size of DV1/KA is probably due to dissociation of NAD+ from the ternary complex [39]. With propan-2-ol and acetone, the kinetic mechanism is steady-state random in both directions [13]. A similar kinetic mechanism probably holds for most branched and secondary alcohols [34]. 5 Pre-steady-state kinetics Pre-steady-state kinetic studies provide the numerical values of the rate constants in the mechanism. The pre-steady-state kinetics of yeast alcohol dehydrogenase has been studied with the help of the KINSIM and FITSIM computer programs of Frieden [42–44]. These computer software packages can simulate the reaction progress curves and calculate the individual rate constants therefrom (Table 4). The magnitudes of the individual rate constants in Scheme 1 were calculated from reaction progress curves in both directions, keeping the concentration of reactants at such a level that dissociation of NAD+ from the central complex was prevented, and therefore excluding the rate constants k11–k14 (Table 4) [45]. 4 Thermodynamics of the yeast alcohol dehydrogenase reaction, at pH 7.0, 25°C [45]   Rate constant  Dissociation constant  ΔG° (kJ/mol)  k1  μM−1 s−1  7±0.2  (11)a  k2/k1  μM  300  −20.08  k2  s−1  2100±57  (3900)a          k4/k3  μM  158 000  (–)a  k41/k31  –  75b  10.70          k42/k32  μM  2110c  −15.26  k9  s−1  3980±97  (4000)a  k10/k9    8.75  5.37  k10  s−1  35 040±870  (35 000)a          k5  s−1  10 900±160  (11 000)a  k5/k6  μM  2180  15.18  k6  μM−1 s−1  5.0±0.04  (4.3)a          k7  s−1  388±5  (480)a  k7/k8  μM  13.80  27.73  k8  μM−1 s−1  28.1±0.5  (44)a                      Total  23.64              Keq=0.000068d  23.7    Rate constant  Dissociation constant  ΔG° (kJ/mol)  k1  μM−1 s−1  7±0.2  (11)a  k2/k1  μM  300  −20.08  k2  s−1  2100±57  (3900)a          k4/k3  μM  158 000  (–)a  k41/k31  –  75b  10.70          k42/k32  μM  2110c  −15.26  k9  s−1  3980±97  (4000)a  k10/k9    8.75  5.37  k10  s−1  35 040±870  (35 000)a          k5  s−1  10 900±160  (11 000)a  k5/k6  μM  2180  15.18  k6  μM−1 s−1  5.0±0.04  (4.3)a          k7  s−1  388±5  (480)a  k7/k8  μM  13.80  27.73  k8  μM−1 s−1  28.1±0.5  (44)a                      Total  23.64              Keq=0.000068d  23.7  aData in parentheses are from the steady-state kinetic measurements of Dickinson and Dickenson [31], at pH 7.0, 25°C. bTaken from Northrop [46]. cCalculated from the equilibrium constant: k4/k3=(k41/k31)/(k42/k32). dCalculated from the Haldane relationship: Keq=V1KiQKp/(V2KiAKB). View Large 4 Thermodynamics of the yeast alcohol dehydrogenase reaction, at pH 7.0, 25°C [45]   Rate constant  Dissociation constant  ΔG° (kJ/mol)  k1  μM−1 s−1  7±0.2  (11)a  k2/k1  μM  300  −20.08  k2  s−1  2100±57  (3900)a          k4/k3  μM  158 000  (–)a  k41/k31  –  75b  10.70          k42/k32  μM  2110c  −15.26  k9  s−1  3980±97  (4000)a  k10/k9    8.75  5.37  k10  s−1  35 040±870  (35 000)a          k5  s−1  10 900±160  (11 000)a  k5/k6  μM  2180  15.18  k6  μM−1 s−1  5.0±0.04  (4.3)a          k7  s−1  388±5  (480)a  k7/k8  μM  13.80  27.73  k8  μM−1 s−1  28.1±0.5  (44)a                      Total  23.64              Keq=0.000068d  23.7    Rate constant  Dissociation constant  ΔG° (kJ/mol)  k1  μM−1 s−1  7±0.2  (11)a  k2/k1  μM  300  −20.08  k2  s−1  2100±57  (3900)a          k4/k3  μM  158 000  (–)a  k41/k31  –  75b  10.70          k42/k32  μM  2110c  −15.26  k9  s−1  3980±97  (4000)a  k10/k9    8.75  5.37  k10  s−1  35 040±870  (35 000)a          k5  s−1  10 900±160  (11 000)a  k5/k6  μM  2180  15.18  k6  μM−1 s−1  5.0±0.04  (4.3)a          k7  s−1  388±5  (480)a  k7/k8  μM  13.80  27.73  k8  μM−1 s−1  28.1±0.5  (44)a                      Total  23.64              Keq=0.000068d  23.7  aData in parentheses are from the steady-state kinetic measurements of Dickinson and Dickenson [31], at pH 7.0, 25°C. bTaken from Northrop [46]. cCalculated from the equilibrium constant: k4/k3=(k41/k31)/(k42/k32). dCalculated from the Haldane relationship: Keq=V1KiQKp/(V2KiAKB). View Large One can see from Table 4 that the magnitudes of rate constants obtained from the computer simulation of reaction progress curves [45] and from the steady-state kinetics [31] are very similar, the differences reflecting only the different enzyme preparations. In the horse liver enzyme, a large conformational change of the enzyme is triggered when the coenzyme binds, well documented both in structural terms [47] and by kinetic methods [48]. Recently, Northrop has reported that moderate pressure increases the capture of benzyl alcohol (V1/KB) in YADH-catalyzed oxidation of this alcohol with NAD+, by activating the hydride transfer step [49]. This means that the collision complex for hydride transfer (*E·NAD+) has a smaller volume than the free alcohol plus the capturing form of the enzyme (E·NAD+) [46]. This was a direct experimental proof for the isomerization step in the yeast enzyme, which enabled the estimation of the equilibrium constant k41/k31[75]; using this value, it was possible to calculate the equilibrium constant k42/k32 (Table 4). Inspection of data in Table 4 clearly shows that, in the forward direction (oxidation of ethanol at neutral pH), the rate-limiting step is not the chemical reaction (k9), but the dissociation of NADH from the EQ-complex (k7). Likewise, NAD+ dissociates much faster from the EA-complex (k2) than NADH dissociates from the EQ-complex (k7). 6 Primary structure YADH-1 is a tetramer, composed of four identical subunits; each subunit consists of a single polypeptide chain with 347 amino acids, with a molecular mass of 36 kDa [47]. Each subunit has one coenzyme-binding site and one firmly bound zinc atom, which is essential for catalysis [50,51]; the catalytic domain provides the ligands to this zinc atom: Cys-46, His-67 and Cys-174. The second zinc atom/subunit is liganded in a tetrahedral arrangement by four sulfur atoms from the cysteine residues 97, 100, 103 and 111; this zinc atom only has a structural role [52]. Table 5 shows the primary structures of the three isoenzymes of YADH [4,53–55]. The alignment of amino acid residues for all 47 members of the ADH family was made progressively rather than pairwise [1]. 5 Primary structure of the three isoenzymes of yeast alcohol dehydrogenase View Large 5 Primary structure of the three isoenzymes of yeast alcohol dehydrogenase View Large 7 The active site The amino acid sequences of horse liver alcohol dehydrogenase and YADH-1 are homologous, and the homology amounts to 25% of the amino acid residues [5]. YADH-1 has been crystallized, but only preliminary crystallographic studies have been reported [56]. The three-dimensional structure of horse liver alcohol dehydrogenase in several binary and ternary complexes with coenzymes, substrates and inhibitors has been solved at high resolution [47]. The tertiary structures of liver and yeast enzyme are highly similar and able to accommodate extensive sequence changes between the enzymes [57]. Analogous to the liver enzyme, the subunits of the yeast enzyme are probably divided into two domains: the catalytic domain and the coenzyme-binding domain. The two domains are unequal in size; the catalytic domain contains 3/5 of all amino acids, whereas the coenzyme-binding domain contains the remaining 2/5 of the amino acids. The domains are separated by a cleft, containing a deep pocket which accommodates the substrate and the nicotinamide moiety of the coenzyme. One domain binds the coenzyme and the other provides ligands to the catalytic zinc, as well as to most of the groups that control substrate specificity [47]. Since the liver and yeast enzymes are homologous, molecular modeling of the yeast enzyme can approximate the structure of one subunit, but not yet the quaternary arrangement [57]. Fig. 1 shows a model of the active site of the yeast enzyme, drawn schematically after a model obtained in a molecular graphics display system by Plapp et al. [58]. The 3D-model of the active site of YADH provides an illustration of the main working machinery of the yeast enzyme. In order to perform catalysis, the active site of the enzyme has to bind a molecule of substrate and a molecule of coenzyme in a productive mode, and, subsequently catalyze a hydride-transfer reaction between them. 1 View largeDownload slide Model of the active site of yeast alcohol dehydrogenase, drawn schematically after Plapp et al. [58]. 1 View largeDownload slide Model of the active site of yeast alcohol dehydrogenase, drawn schematically after Plapp et al. [58]. The adenosine-binding site is easily accessible from solution, whereas the nicotinamide-binding site is situated at the center of the molecule, buried deep inside the protein [47]. Numerous amino acid residues in the primary structure of the enzyme are involved in substrate and coenzyme binding and in catalysis (Table 6). 6 Positions of residues that participate in enzymatic functions of the yeast enzyme (adopted from Jörnval et al. [57]) Adenine binding pocket  Nicotinamide  Interior    Thr-178    Ser-198  Ile-250      Ile-222  Ser-269  Substrate-binding pocket  Gly-224  Ala-274  Trp-57  Thr-141  Phe-243  Ala-277  Trp-93  Met-294  Surface    Asn-110  Ala-296  Ser-271  Ala-273  Leu-132  Ile-318      Tyr-140    Adenosine–ribose binding      Gly-199  Lys-228  Proton relay system  Asp-223  Ser-269  Thr-48  His-51  Gly-225            Ligands to active-site zinc atom  Pyrophosphate binding  Inner sphere    His-47  Leu-203  Cys-46  Cys-174  Gly-202    His-67        Second sphere    Nicotinamide ribose  Asp-49  Glu-68  Gly-293  Ser-269      Adenine binding pocket  Nicotinamide  Interior    Thr-178    Ser-198  Ile-250      Ile-222  Ser-269  Substrate-binding pocket  Gly-224  Ala-274  Trp-57  Thr-141  Phe-243  Ala-277  Trp-93  Met-294  Surface    Asn-110  Ala-296  Ser-271  Ala-273  Leu-132  Ile-318      Tyr-140    Adenosine–ribose binding      Gly-199  Lys-228  Proton relay system  Asp-223  Ser-269  Thr-48  His-51  Gly-225            Ligands to active-site zinc atom  Pyrophosphate binding  Inner sphere    His-47  Leu-203  Cys-46  Cys-174  Gly-202    His-67        Second sphere    Nicotinamide ribose  Asp-49  Glu-68  Gly-293  Ser-269      View Large 6 Positions of residues that participate in enzymatic functions of the yeast enzyme (adopted from Jörnval et al. [57]) Adenine binding pocket  Nicotinamide  Interior    Thr-178    Ser-198  Ile-250      Ile-222  Ser-269  Substrate-binding pocket  Gly-224  Ala-274  Trp-57  Thr-141  Phe-243  Ala-277  Trp-93  Met-294  Surface    Asn-110  Ala-296  Ser-271  Ala-273  Leu-132  Ile-318      Tyr-140    Adenosine–ribose binding      Gly-199  Lys-228  Proton relay system  Asp-223  Ser-269  Thr-48  His-51  Gly-225            Ligands to active-site zinc atom  Pyrophosphate binding  Inner sphere    His-47  Leu-203  Cys-46  Cys-174  Gly-202    His-67        Second sphere    Nicotinamide ribose  Asp-49  Glu-68  Gly-293  Ser-269      Adenine binding pocket  Nicotinamide  Interior    Thr-178    Ser-198  Ile-250      Ile-222  Ser-269  Substrate-binding pocket  Gly-224  Ala-274  Trp-57  Thr-141  Phe-243  Ala-277  Trp-93  Met-294  Surface    Asn-110  Ala-296  Ser-271  Ala-273  Leu-132  Ile-318      Tyr-140    Adenosine–ribose binding      Gly-199  Lys-228  Proton relay system  Asp-223  Ser-269  Thr-48  His-51  Gly-225            Ligands to active-site zinc atom  Pyrophosphate binding  Inner sphere    His-47  Leu-203  Cys-46  Cys-174  Gly-202    His-67        Second sphere    Nicotinamide ribose  Asp-49  Glu-68  Gly-293  Ser-269      View Large 7.1 Substrate-binding pocket The inner wall of the pocket is lined with hydrophobic side chains from the residues Trp-57, Trp-93, Asn-110, Leu-132, Tyr-140, Thr-141, Met-294, Ala-296, and Ile-318, which are from the same subunit as the zinc ligands. The substrate-binding site near zinc is narrow, because access is limited by Trp-93 and Thr-48. The voluminous amino acid side chains of Trp-57, Trp-93 and Met-294 make the substrate-binding pocket in the yeast enzyme much more narrow than the corresponding pocket in the horse liver enzyme. 7.2 Ligands to the active-site zinc At the bottom of the substrate-binding pocket, a zinc atom is coordinated to three protein ligands: two thiolates from Cys-46 and Cys-174, and one imidazole nitrogen of His-67. The other imidazole nitrogen of His-67 is hydrogen-bonded to a carboxylic group of Asp-49. A carboxylic group of Glu-68 is also in close proximity to the active-site zinc atom. Asp-49 and Glu-68 are the residues conserved in all known zinc-dependent alcohol dehydrogenases; instead of being inner-shpere ligands to the zinc, both amino acids are situated in the second sphere. The only polar groups in the pocket close to zinc are the zinc ligands, the nicotinamide moiety of the coenzyme, and the side chain of Thr-48. 7.3 Nicotinamide ring The nicotinamide ring binds in a cleft in the interior of the protein, close to the center of the molecule. On one side the ring interacts with Thr-178, Leu-203 and Met-294. The other side faces the active site, and is close to the catalytic zinc atom and the sulfur ligands of Cys-46 and Cys-174. The oxygen atom of the carboxamide group is hydrogen-bonded to the main-chain nitrogen atom of Val-319. The nitrogen atom of the carboxamide group is hydrogen-bonded to the carboxyl oxygens of Val-292 and Ser-317. The side chain of Thr-178 helps to keep the nicotinamide ring of the nucleotide in the correct stereochemical position for hydride transfer (Fig. 5); Thr-178 is conserved in all known homologous alcohol dehydrogenases. 5 View largeDownload slide Stereospecificity of YADH catalysis. NADH binds anti, presenting Re-hydrogen (HRe) to acetaldehyde lying above the coenzyme in this diagram. For clarity, Thr-178 is not shown; the methyl group of this side chain lies below and to the left of the nicotinamide behind Leu-203 (reproduced from Weinhold et al. [64], with permission of the corresponding author). 5 View largeDownload slide Stereospecificity of YADH catalysis. NADH binds anti, presenting Re-hydrogen (HRe) to acetaldehyde lying above the coenzyme in this diagram. For clarity, Thr-178 is not shown; the methyl group of this side chain lies below and to the left of the nicotinamide behind Leu-203 (reproduced from Weinhold et al. [64], with permission of the corresponding author). 7.4 The proton-relay system It was proposed by Eklund et al. [59] that the hydrogen-bonded relay system in the liver enzyme (Fig. 3),   stretching from His-51 on the surface of the enzyme to the active-site zinc atom in the interior of the enzyme, serves as a proton conductor which helps the dissociation of alcohol to alcoholate in the productive ternary enzyme·NAD+·alcohol-complex. The yeast enzyme has the same proton relay system with, however, Ser substituted with Thr (Fig. 1) [58]. Therefore, since the enzyme·NAD+·alcoholate-complex is considered a true transition state in the yeast enzyme catalysis [60], the proton relay system must greatly accelerate the same. 3 View largeDownload slide The chemical mechanism of action of alcohol dehydrogenase [36]. 3 View largeDownload slide The chemical mechanism of action of alcohol dehydrogenase [36]. 7.5 Binding of the coenzyme The coenzyme is bound to the apoenzyme by numerous secondary valence forces. Important amino acid residues are: Asp-223, which is hydrogen-bounded to AMP-ribose, His-47, forming a salt bridge with AMP-orthophosphate, and Leu-203, forming a hydrogen bond to NMN-orthophosphate. 8 Mutations in the yeast enzyme The three yeast ADH genes have been cloned and described [4,54,55]. Therefore, it was possible to change individual amino acids in the primary structure of YADH-1 via site-directed mutagenesis and isolate a quantity of mutated enzymes (Table 7). 7 Steady-state kinetic constants for YADH mutants, with ethanol and acetaldehyde as substrates, determined at pH 7.3, 30°C Mutant  V1 (s−1)  V1/KA (mM−1 s−1)  V1/KB (mM−1 s−1)  V2 (s−1)  V2/KQ (mM−1 s−1)  V2/KP (mM−1 s−1)  Source  YADH-1  340  2000  20  1700  15 500  1545  [9]  Substrate-binding pocket  Met294Leu  500  794  26.3  2100  26 250  2100  [9]  Trp57Met  220  265  4.9  1900  6 790  513  [20]  Trp57Leua  99  91  7.4  211  2 245  ND  [19]  Trp93Ala  110  48  0.07  ND  ND  ND  [20]  Ligands to the active-site zinc  Asp49Asn  7.5  0.83  0.02  113  125  2.3  [39]  Glu68Gln  9.9  24  0.24  730  4 560  13  [39]  The proton relay system  Thr48Ser  200  2200  11.8  1500  13 640  2027  [20]  Thr48Ser:Trp93Ala  140  152  0.033  530  4 077  5.7  [20]  Thr48Ser:Trp57Met:Trp93Ala  120  22.2  0.75  ND  ND  ND  [20]  Thr48Cys  <1  No detectable activity  [58]  Thr48Ala  <1  No detectable activity  [58]  His51Gln  27  245  1.4  2800  25 450  215  [61]  His51Glu  2  26  0.26  ND  ND  ND  [58]  Mutant  V1 (s−1)  V1/KA (mM−1 s−1)  V1/KB (mM−1 s−1)  V2 (s−1)  V2/KQ (mM−1 s−1)  V2/KP (mM−1 s−1)  Source  YADH-1  340  2000  20  1700  15 500  1545  [9]  Substrate-binding pocket  Met294Leu  500  794  26.3  2100  26 250  2100  [9]  Trp57Met  220  265  4.9  1900  6 790  513  [20]  Trp57Leua  99  91  7.4  211  2 245  ND  [19]  Trp93Ala  110  48  0.07  ND  ND  ND  [20]  Ligands to the active-site zinc  Asp49Asn  7.5  0.83  0.02  113  125  2.3  [39]  Glu68Gln  9.9  24  0.24  730  4 560  13  [39]  The proton relay system  Thr48Ser  200  2200  11.8  1500  13 640  2027  [20]  Thr48Ser:Trp93Ala  140  152  0.033  530  4 077  5.7  [20]  Thr48Ser:Trp57Met:Trp93Ala  120  22.2  0.75  ND  ND  ND  [20]  Thr48Cys  <1  No detectable activity  [58]  Thr48Ala  <1  No detectable activity  [58]  His51Gln  27  245  1.4  2800  25 450  215  [61]  His51Glu  2  26  0.26  ND  ND  ND  [58]  ND=not determined. aDetermined at pH 8.2, 25°C. View Large 7 Steady-state kinetic constants for YADH mutants, with ethanol and acetaldehyde as substrates, determined at pH 7.3, 30°C Mutant  V1 (s−1)  V1/KA (mM−1 s−1)  V1/KB (mM−1 s−1)  V2 (s−1)  V2/KQ (mM−1 s−1)  V2/KP (mM−1 s−1)  Source  YADH-1  340  2000  20  1700  15 500  1545  [9]  Substrate-binding pocket  Met294Leu  500  794  26.3  2100  26 250  2100  [9]  Trp57Met  220  265  4.9  1900  6 790  513  [20]  Trp57Leua  99  91  7.4  211  2 245  ND  [19]  Trp93Ala  110  48  0.07  ND  ND  ND  [20]  Ligands to the active-site zinc  Asp49Asn  7.5  0.83  0.02  113  125  2.3  [39]  Glu68Gln  9.9  24  0.24  730  4 560  13  [39]  The proton relay system  Thr48Ser  200  2200  11.8  1500  13 640  2027  [20]  Thr48Ser:Trp93Ala  140  152  0.033  530  4 077  5.7  [20]  Thr48Ser:Trp57Met:Trp93Ala  120  22.2  0.75  ND  ND  ND  [20]  Thr48Cys  <1  No detectable activity  [58]  Thr48Ala  <1  No detectable activity  [58]  His51Gln  27  245  1.4  2800  25 450  215  [61]  His51Glu  2  26  0.26  ND  ND  ND  [58]  Mutant  V1 (s−1)  V1/KA (mM−1 s−1)  V1/KB (mM−1 s−1)  V2 (s−1)  V2/KQ (mM−1 s−1)  V2/KP (mM−1 s−1)  Source  YADH-1  340  2000  20  1700  15 500  1545  [9]  Substrate-binding pocket  Met294Leu  500  794  26.3  2100  26 250  2100  [9]  Trp57Met  220  265  4.9  1900  6 790  513  [20]  Trp57Leua  99  91  7.4  211  2 245  ND  [19]  Trp93Ala  110  48  0.07  ND  ND  ND  [20]  Ligands to the active-site zinc  Asp49Asn  7.5  0.83  0.02  113  125  2.3  [39]  Glu68Gln  9.9  24  0.24  730  4 560  13  [39]  The proton relay system  Thr48Ser  200  2200  11.8  1500  13 640  2027  [20]  Thr48Ser:Trp93Ala  140  152  0.033  530  4 077  5.7  [20]  Thr48Ser:Trp57Met:Trp93Ala  120  22.2  0.75  ND  ND  ND  [20]  Thr48Cys  <1  No detectable activity  [58]  Thr48Ala  <1  No detectable activity  [58]  His51Gln  27  245  1.4  2800  25 450  215  [61]  His51Glu  2  26  0.26  ND  ND  ND  [58]  ND=not determined. aDetermined at pH 8.2, 25°C. View Large In recent years, a number of genetically engineered mutants of YADH-1 were isolated and kinetically characterized, principally by Plapp and his co-workers. Most of these mutations involve amino acids which are intimately involved in the binding of substrates and in catalysis, and provide information about the general principles concerning the function of the catalytic residues. Table 7 shows the steady-state kinetic properties of all YADH mutants described so far participating in substrate binding and in catalysis. 8.1 Substrate-binding pocket (Met-294, Trp-57, Trp-93) An exchange of Leu for Met-294, on the edge of the substrate-binding pocket, has very little influence on the steady-state kinetic properties of the enzyme with ethanol or acetaldehyde. On the other hand, the Met294Leu mutant has a 10-fold lower catalytic activity (V1) with butan-1-ol, indicating that the C4-atom of butan-1-ol is in a close contact with Met-294, whereas the shorter ethanol is not [9]. An exchange of Met or Leu for Trp-57 decreases the catalytic efficiency (V1/KB) with ethanol only three- to four-fold, whereas an exchange of Ala for Trp-93 decreases the catalytic efficiency 300-fold; with an enlargement of the substrate-binding pocket in the latter case, the enzyme acquires weak activity with branched-chain alcohols (2-methyl-1-butanol, 3-methyl-1-butanol) and benzyl alcohol [19,20]. 8.2 Ligands to the active-site zinc (Asp-49, Glu-68) The carboxylate group of Asp-49 is hydrogen-bonded to His-67, which in turn coordinates the active-site zinc; in addition, the carboxylate group of Glu-68 is in the vicinity of the active-site zinc. If Asn is substituted for Asp-49 or Gln for Glu-68, a negative charge is removed from the vicinity of the active-site zinc; these substitutions reduce the catalytic efficiency with ethanol (V1/KB) 1000 times and 100 times, respectively, and the catalytic constant (V1) 40 times. These reductions in activity were interpreted by an increased electrostatic potential near the active-site zinc, due to removal of negative charges; as a consequence the activity is decreased by hindering isomerizations of enzyme–substrate complexes [39]. 8.3 The proton-relay system (Thr-48, His-51) An exchange of Ser for Thr-48 does not interrupt the hydrogen bonding in the proton relay system and, as expected, the activity of the Thr48Ser mutant is very similar to that of the wild-type. The double mutant Thr48Ser:Trp93Ala and the triple mutant Thr48Ser:Trp57Met:Trp93Ala show decreased activities that are obviously due to removal of bulky tryptophan residues from the substrate-binding pocket [20]. An exchange of Cys or Ala for Thr-48 disrupts the hydrogen bonding in the relay system and, as expected, renders the enzyme inactive [58]. The role of His-51 in catalysis has been tested by replacing it with glutamine or glutamic acid [58,61]. These residues have an appropriate size to form the hydrogen bond with the 2′-hydroxyl group of the nicotinamide ribose; thus, binding of the coenzyme in the mutant enzymes could resemble binding in the wild-type enzyme. On the other hand, a glutamine residue would not be able to participate in base catalysis, whereas a glutamate residue could accept a proton. Plapp et al. [58] have found that a wild-type enzyme has a distinct pKa value of 7.7 in the pH-profile for the V1/KB function. Replacement of His-51 with Gln or Glu reduces the value of V1/KB 13-fold and 60-fold at pH 7.3, respectively; in addition, the pKa value of 7.7 in the pH profile of the V1/KB function is abolished in both cases. These results were interpreted by a mechanism in which the amino acid residue in the mutant enzyme hinders the deprotonation of alcohol through the proton relay system [58]. On that interpretation, these results are consistent with the role of His-51 in the proton relay system, where it participates as a base. 9 Binding of coenzymes Fig. 2 summarizes the steady-state and ligand-binding data relevant for the binding of coenzymes to the free enzyme. 2 View largeDownload slide pH profiles for the binding parameters of coenzymes to the free enzyme; rate constants k7 and k8, as in Scheme 1 (adopted from Leskovac et al. [34]). 2 View largeDownload slide pH profiles for the binding parameters of coenzymes to the free enzyme; rate constants k7 and k8, as in Scheme 1 (adopted from Leskovac et al. [34]). The dissociation constant of the E·NAD+ complex for the yeast enzyme, KE,NAD+, is practically pH-independent; on the other hand, the dissociation constant of the E·NADH complex, KE,NADH, decreases with lower pH-value over three apparent pKa values (6.6, 8.0 and 9.0). The association rate constant for the binding of NADH to the free enzyme (k8) decreases in alkaline over a single pKa value 7.8, while the dissociation rate constant for the E·NADH-complex (k7) is almost pH-independent, from pH 6.5 to 9.0. In recent years, a number of genetically engineered mutants of YADH-I, with mutations in the adenylate-binding pocket, have been isolated and kinetically characterized, principally by Plapp and his co-workers (Table 8). The following general conclusions may be drawn from the kinetic data shown in Table 8. 8 Steady-state kinetic constants for YADH mutants in the adenylate binding pocket, with ethanol and acetaldehyde as substrates, determined at pH 7.3, 30°C Mutant  V1 (s−1)  V1/KA (mM−1 s−1)  V1/KB (mM−1 s−1  V2 (s−1)  V2/KQ (mM−1 s−1)  V2/KP (mM−1 s−1)  Source  YADH-1  340  2000  20  1700  15 500  1 545  [9]  Adenine site substitutions  Ser198Phe  40  1.25  0.14  150  25  71  [62]  Gly224Ile  360  414  16  4000  6 060  20 000  [62]  Gly225Arg  550  1222  21  2400  12 000  18 000  [62]  Adenosine–ribose binding  Asp223Gly  38  2.1  0.2  300  60  75  [63]  Asp223Gly:Gly225Arg  17  0.94  0.13  110  18.33  20  [63]  Pyrophosphate binding  Leu203Alaa  106  56.4  ND  ND  ND  ND  [64]  Leu203Ala:Thr178Sera  31.9  61.3  ND  ND  ND  ND  [64]  His47Arg  60  400  0.9  460  46 000  98  [32]  Gly204Ala  8  0.26  0.02  200  25  11  [62]  Ala200Δ:Ala201Leub  67  13.13  1.4  ND  ND  ND  [62]  NMN–ribose binding  Ser269Ile  1.0  0.36  0.003  5.4  13.85  0.31  [62]  Mutant  V1 (s−1)  V1/KA (mM−1 s−1)  V1/KB (mM−1 s−1  V2 (s−1)  V2/KQ (mM−1 s−1)  V2/KP (mM−1 s−1)  Source  YADH-1  340  2000  20  1700  15 500  1 545  [9]  Adenine site substitutions  Ser198Phe  40  1.25  0.14  150  25  71  [62]  Gly224Ile  360  414  16  4000  6 060  20 000  [62]  Gly225Arg  550  1222  21  2400  12 000  18 000  [62]  Adenosine–ribose binding  Asp223Gly  38  2.1  0.2  300  60  75  [63]  Asp223Gly:Gly225Arg  17  0.94  0.13  110  18.33  20  [63]  Pyrophosphate binding  Leu203Alaa  106  56.4  ND  ND  ND  ND  [64]  Leu203Ala:Thr178Sera  31.9  61.3  ND  ND  ND  ND  [64]  His47Arg  60  400  0.9  460  46 000  98  [32]  Gly204Ala  8  0.26  0.02  200  25  11  [62]  Ala200Δ:Ala201Leub  67  13.13  1.4  ND  ND  ND  [62]  NMN–ribose binding  Ser269Ile  1.0  0.36  0.003  5.4  13.85  0.31  [62]  ND=not determined. aDetermined at pH 8.2, 25°C. bAlignment of amino acids according to Table 5. Ala200 is an insertion in the yeast enzyme with respect to other members of the alcohol dehydrogenase family of enzymes; therefore, this residue is not counted in the primary structure that follows after this residue [1]. View Large 8 Steady-state kinetic constants for YADH mutants in the adenylate binding pocket, with ethanol and acetaldehyde as substrates, determined at pH 7.3, 30°C Mutant  V1 (s−1)  V1/KA (mM−1 s−1)  V1/KB (mM−1 s−1  V2 (s−1)  V2/KQ (mM−1 s−1)  V2/KP (mM−1 s−1)  Source  YADH-1  340  2000  20  1700  15 500  1 545  [9]  Adenine site substitutions  Ser198Phe  40  1.25  0.14  150  25  71  [62]  Gly224Ile  360  414  16  4000  6 060  20 000  [62]  Gly225Arg  550  1222  21  2400  12 000  18 000  [62]  Adenosine–ribose binding  Asp223Gly  38  2.1  0.2  300  60  75  [63]  Asp223Gly:Gly225Arg  17  0.94  0.13  110  18.33  20  [63]  Pyrophosphate binding  Leu203Alaa  106  56.4  ND  ND  ND  ND  [64]  Leu203Ala:Thr178Sera  31.9  61.3  ND  ND  ND  ND  [64]  His47Arg  60  400  0.9  460  46 000  98  [32]  Gly204Ala  8  0.26  0.02  200  25  11  [62]  Ala200Δ:Ala201Leub  67  13.13  1.4  ND  ND  ND  [62]  NMN–ribose binding  Ser269Ile  1.0  0.36  0.003  5.4  13.85  0.31  [62]  Mutant  V1 (s−1)  V1/KA (mM−1 s−1)  V1/KB (mM−1 s−1  V2 (s−1)  V2/KQ (mM−1 s−1)  V2/KP (mM−1 s−1)  Source  YADH-1  340  2000  20  1700  15 500  1 545  [9]  Adenine site substitutions  Ser198Phe  40  1.25  0.14  150  25  71  [62]  Gly224Ile  360  414  16  4000  6 060  20 000  [62]  Gly225Arg  550  1222  21  2400  12 000  18 000  [62]  Adenosine–ribose binding  Asp223Gly  38  2.1  0.2  300  60  75  [63]  Asp223Gly:Gly225Arg  17  0.94  0.13  110  18.33  20  [63]  Pyrophosphate binding  Leu203Alaa  106  56.4  ND  ND  ND  ND  [64]  Leu203Ala:Thr178Sera  31.9  61.3  ND  ND  ND  ND  [64]  His47Arg  60  400  0.9  460  46 000  98  [32]  Gly204Ala  8  0.26  0.02  200  25  11  [62]  Ala200Δ:Ala201Leub  67  13.13  1.4  ND  ND  ND  [62]  NMN–ribose binding  Ser269Ile  1.0  0.36  0.003  5.4  13.85  0.31  [62]  ND=not determined. aDetermined at pH 8.2, 25°C. bAlignment of amino acids according to Table 5. Ala200 is an insertion in the yeast enzyme with respect to other members of the alcohol dehydrogenase family of enzymes; therefore, this residue is not counted in the primary structure that follows after this residue [1]. View Large 9.1 Adenine site substitutions (Ser-198, Gly-224, Gly-225) Gly224Ile and Gly225Arg mutants have only modest effects on coenzyme binding and other kinetic constants, but the Ser198Phe mutant significantly decreases its affinity for coenzymes and turnover numbers. 9.2 Adenosine–ribose binding (Asp-223) The Asp223Gly:Gly225Arg double mutant shows a decrease in all kinetic parameters, but uses NAD(H) and NADP(H) with about the same efficiency. 9.3 Pyrophosphate binding (His-47, Ala-200, Leu-203, Gly-204) Mutation of the residues Ala-200, Leu-203 or Gly-204 decreases all kinetic parameters significantly, suggesting that these amino acids are essential for the binding of the pyrophosphate moiety of the coenzyme. On the other hand, substitution of His-47 by the basic amino acid Arg decreases the catalytic activity with NAD(H) only modestly. 9.4 Nicotinamide–ribose binding (Ser-269) The Ser269Ile mutant decreases its turnover numbers by 350-fold. Studies of the mutants in the adenylate-binding site of the enzyme show that several amino acid residues at the proposed adenylate-binding site of the enzyme are important for coenzyme binding and formation of productive ternary complexes. The Asp223Gly:Gly225Arg double mutant was the only mutant that uses NAD(H) and NADP(H) with about the same efficiency; this result suggests that conversion of the coenzyme specifically requires multiple substitutions [62]. Mutations of amino acids Leu-203 and Thr-178 have been performed in order to locate the structural determinants of the high stereospecificity of the enzyme for the coenzyme NAD(H) [64]. 10 Chemical mechanism Primary structure, tertiary structure and point mutations in the yeast enzyme, outlined in the preceding sections, strongly suggest that the integrity of the proton relay system is indispensable for the activity of the enzyme. Based on this integrity of the relay system, which is maintained throughout the catalytic cycle, Cook and Cleland [60] have proposed the chemical mechanism of action for the yeast enzyme as shown in Scheme 2. Scheme 2 View largeDownload slide Scheme 2 View largeDownload slide In this mechanism, B and P represent alcohol and ketone, and k3, k4, k5 and k6 represent hydride-transfer steps; X is an intermediate with the stoichiometry of an alkoxide, and k1 and k2 are the steps in which a proton is transferred from B to a group on the enzyme to give X, and similarly for the reverse process. An assignment of appropriate pKa values to all dissociation forms of the enzyme in Scheme 2 was founded on studies of the pH dependence of the steady-state kinetics and ligand-binding parameters [14,16,26,33–36,65], as outlined below. Table 9 shows the macroscopic pKa values calculated from the pH profiles of the maximal rates (V1) and the specificity constants (V/K) with various substrates. 9 Macroscopic pKa values and pH-independent limiting constants in various YADH-catalyzed reactions (adopted from Leskovac et al. [36]) Substrate  pKa  Limiting constant  Dixon–Webb plot  Butan-1-ol  6.1  191  increases with pH  V1 (s−1)  7.3        8.3      Propan-2-ol  6.2  81  increases with pH  V1/KB (s−1)  7.4        8.3      Propan-1-ol  6.7  9.0  increases with pH  V1/KB (mM−1 s−1)  7.4        8.2      Propan-2-ol  6.5  155  increases with pH  V1/KB (M−1 s−1)  7.1        7.8      Acetone  7.9  6.9  decreases with pH  V1/KB (M−1 s−1)  8.2        9.0      DACA  8.0  0.25  decreases with pH  V2/KP (mM−1 s−1)        NDMAa  8.0  2.2  plateau at low pH  V2/KP (mM−1 s−1)    0.9  plateau at high pH  Substrate  pKa  Limiting constant  Dixon–Webb plot  Butan-1-ol  6.1  191  increases with pH  V1 (s−1)  7.3        8.3      Propan-2-ol  6.2  81  increases with pH  V1/KB (s−1)  7.4        8.3      Propan-1-ol  6.7  9.0  increases with pH  V1/KB (mM−1 s−1)  7.4        8.2      Propan-2-ol  6.5  155  increases with pH  V1/KB (M−1 s−1)  7.1        7.8      Acetone  7.9  6.9  decreases with pH  V1/KB (M−1 s−1)  8.2        9.0      DACA  8.0  0.25  decreases with pH  V2/KP (mM−1 s−1)        NDMAa  8.0  2.2  plateau at low pH  V2/KP (mM−1 s−1)    0.9  plateau at high pH  View Large 9 Macroscopic pKa values and pH-independent limiting constants in various YADH-catalyzed reactions (adopted from Leskovac et al. [36]) Substrate  pKa  Limiting constant  Dixon–Webb plot  Butan-1-ol  6.1  191  increases with pH  V1 (s−1)  7.3        8.3      Propan-2-ol  6.2  81  increases with pH  V1/KB (s−1)  7.4        8.3      Propan-1-ol  6.7  9.0  increases with pH  V1/KB (mM−1 s−1)  7.4        8.2      Propan-2-ol  6.5  155  increases with pH  V1/KB (M−1 s−1)  7.1        7.8      Acetone  7.9  6.9  decreases with pH  V1/KB (M−1 s−1)  8.2        9.0      DACA  8.0  0.25  decreases with pH  V2/KP (mM−1 s−1)        NDMAa  8.0  2.2  plateau at low pH  V2/KP (mM−1 s−1)    0.9  plateau at high pH  Substrate  pKa  Limiting constant  Dixon–Webb plot  Butan-1-ol  6.1  191  increases with pH  V1 (s−1)  7.3        8.3      Propan-2-ol  6.2  81  increases with pH  V1/KB (s−1)  7.4        8.3      Propan-1-ol  6.7  9.0  increases with pH  V1/KB (mM−1 s−1)  7.4        8.2      Propan-2-ol  6.5  155  increases with pH  V1/KB (M−1 s−1)  7.1        7.8      Acetone  7.9  6.9  decreases with pH  V1/KB (M−1 s−1)  8.2        9.0      DACA  8.0  0.25  decreases with pH  V2/KP (mM−1 s−1)        NDMAa  8.0  2.2  plateau at low pH  V2/KP (mM−1 s−1)    0.9  plateau at high pH  View Large Table 10 presents the pKa values calculated from the pH profiles of binding constants (Ki) for competitive dead-end inhibitors. 10 Macroscopic pKa values and pH-independent constants for ternary complexes of YADH with competitive dead-end inhibitorsa Complex  pKa  Limiting constant  E·NAD++Az⇌E·NAD+·Az  7.9  0.95 mM (at low pH)  E·NADH+AA⇌E·NADH·AA  8.3  45.8 mM (low pH)      118 mM (high pH)  Complex  pKa  Limiting constant  E·NAD++Az⇌E·NAD+·Az  7.9  0.95 mM (at low pH)  E·NADH+AA⇌E·NADH·AA  8.3  45.8 mM (low pH)      118 mM (high pH)  aCalculated from the data of Leskovac et al. [35]. View Large 10 Macroscopic pKa values and pH-independent constants for ternary complexes of YADH with competitive dead-end inhibitorsa Complex  pKa  Limiting constant  E·NAD++Az⇌E·NAD+·Az  7.9  0.95 mM (at low pH)  E·NADH+AA⇌E·NADH·AA  8.3  45.8 mM (low pH)      118 mM (high pH)  Complex  pKa  Limiting constant  E·NAD++Az⇌E·NAD+·Az  7.9  0.95 mM (at low pH)  E·NADH+AA⇌E·NADH·AA  8.3  45.8 mM (low pH)      118 mM (high pH)  aCalculated from the data of Leskovac et al. [35]. View Large The specificity constants V/K with ‘nonsticky’ substrates, such as propan-1-ol, propan-2-ol, NDMA, DACA and acetone, provide information on catalytically active groups in enzyme–coenzyme complexes [66], if the pH profiles of V/K are fitted to initial-rate equations appropriate to the mechanism in Scheme 2[36]. In this way, the pK1 (8.0) and pK5 (7.9–8.0) values in Scheme 2 were estimated. From the binding of azide, a dead-end inhibitor competitive with alcohols, the value for pK1 (7.9) was confirmed; from the binding of acetamide, a dead-end inhibitor competitive with aldehydes, the values for pK4 (8.3) and pK5 (7.9) were estimated. pH profiles for the V1 function provide information on catalytically active groups in the productive ternary enzyme·NAD+·alcohol-complex [66]. In this way the pK2 value was estimated (8.3), from the pH profiles of V1 with butan-1-ol and propan-2-ol. An indirect estimation provided the value of pK3 (8.3) [36]. The chemical mechanism of action, presented in Scheme 1, can be drawn entirely in terms of the proton relay system, as is shown in Fig. 3; in Fig. 3, however, the Thr-48 residue was omitted from the relay for the sake of simplicity. The key feature of Fig. 3 is that His-51 lies at the surface of the protein and thus can be deprotonated as in the conversion of HEAX to EAX or HEQP to EPQ, while reactants are bound and the state of protonation of molecules in the substrate-binding site is locked. Thus, HEAX can be deprotonated to EAX without preventing subsequent hydride transfer. A different view on the chemical mechanism of action of yeast alcohol dehydrogenase has been presented by Brändén et al. [5]. These authors proposed that the Zn2+-bound water dissociates when the coenzyme NAD+ is added; the remaining (OH)− deprotonates the alcohol, which is then bound to the Zn2+ ion as the fourth ligand. Fig. 4 shows this dissociation in the proton relay system. Recently, Nadolny and Zundel [67] have claimed experimental evidence supporting the above mechanism. These authors obtained Fourier-transform infrared (FTIR) spectra of various complexes of yeast alcohol dehydrogenase with NAD+ and coenzyme analogs; from the FTIR spectra they concluded that, upon binding of NAD+ to the enzyme, N1 of the coenzyme adenosine becomes protonated and the molecule of water in the active site dissociates to a hydroxyl anion. It was postulated that the positive charge is conducted from the zinc-bound water to histidine-51 and then further to the N1-atom of the adenine rest via the proton relay system through the protein. Thus the binding of NAD+ to the enzyme shifts the equilibrium 1→2 in Fig. 4 to the right. The substrate, alcohol, is then deprotonated by the (OH)− bound to the Zn2+ ion and forms the structure 3. The experiments of Nadolny and Zundel [67] with the yeast enzyme were conducted at pH 7.5 and they do not explain the pH dependence of the enzyme activity. Further, the proposed mechanism lacks the explanation for the conductance of the positive charge from His-51 to adenine across a distance of approximately 7 Å. 11 Hydride transfer One of the classical aspects of coenzyme binding to yeast alcohol dehydrogenase is the A-stereospecificity of the coenzyme [68]. YADH-catalyzed reactions are highly stereospecific; the enzyme catalyzes the transfer of the Re-hydrogen (pro-R or A-type) at the 4-position of NADH to the carbonyl carbon of the substrate (Fig. 5). The stereochemical fidelity of the hydride transfer reaction is very high, and YADH makes but one stereochemical ‘mistake’ every 7 000 000 turnovers. If the bulky side chain of Leu-203 is exchanged with Ala, the Leu203Ala mutant (Table 7) makes one stereochemical ‘mistake’ every 850 000 turnovers with NADH, and every 450 turnovers with thio-NADH, which has a weaker hydrogen-bonding capacity. From this, it was concluded that the decrease in stereochemical fidelity comes from an increase in the transfer rate of the 4-Si-hydrogen of NADH. The nicotinamide ring of the coenzyme is kept in a correct position for hydride transfer mainly by hydrogen bonds between its amide group and Val-292 and Val-319, and the rotation of 180° around the glycosidic bond is obstructed mainly by the side chain of Leu-203 [64]. The main reaction catalyzed by alcohol dehydrogenase is, in principle, a very simple reaction. An alcohol group is oxidized by the removal of a proton from the hydroxyl group and by the transfer of a hydride ion from the adjacent carbon atom to NAD+. By analogy with the horse liver enzyme [47], we may assume that hydride transfer in the yeast enzyme occurs in a completely water-free environment. Direct transfer of a hydride ion is facilitated in a hydrophobic environment, where water is excluded. The positive charge on the nicotinamide ring is crucial for the enhanced binding of alcohol to the enzyme; insertion of the positive charge in this hydrophobic environment facilitates formation of the negatively charged alcoholate ion. The creation of an alcoholate ion greatly facilitates hydride transfer. The important role of the zinc atom in alcohol oxidation is to stabilize the alcoholate ion for the hydride-transfer step. In the reverse direction, zinc functions as an electron attractor, which gives rise to an increased electrophilic character of the aldehyde, consequently facilitating the transfer of a hydride ion to the aldehyde. Thus, the proposed mechanism is essentially electrophilic catalysis mediated by the active-site zinc atom. The overall oxidation of alcohol to aldehyde involves the net release of one proton (Eq. 1); the ultimate source of this proton is alcohol. The release of a proton from the bound alcohol occurs in the center of the enzyme molecule in a region that is inaccessible to solution; the proton is transferred by certain groups on the enzyme to the surrounding solution (Fig. 1). Because water is not directly involved in the catalytic reaction, that is, no hydrolysis or hydration, there is no reason to suggest a role for a water molecule at the active site of YADH [36]. In catalysis, the molecules of the substrate and the nicotinamide ring of the coenzyme probably do not have fixed positions. The rearrangement of electron configuration on the carbon atom from the sp2 hybridization in aldehyde to the sp3 in alcohol, requires different pathways for hydride transfer and, consequently, different relative orientations [69]. Primary and secondary kinetic isotope effects (kH/kD, kH/kT and kD/kT) in YADH-catalyzed reactions have been studied as a probe of quantum-mechanical hydrogen tunneling. Hydrogen tunneling was first suggested in YADH-catalyzed reactions following the measurement of anomalously large secondary kinetic isotope effects [70]. In the absence of tunneling and coupled motion, a secondary kinetic isotope effect is expected to be intermediate between unity and the value of the equilibrium isotope effect [71]. Thus, Cook et al. [70] studied the oxidation of 2-propanol with [4-2H]NAD+,   7 and found a secondary kinetic isotope effect of 1.22 and an equilibrium isotope effect of 0.89, which is indicative of hydrogen tunneling. In hydride-transfer reactions catalyzed by YADH, one can measure the kH/kD, kH/kT, and kD/kT primary, and, similarly, the secondary isotope effects. The Swain–Schaad relationship states that, in the absence of tunneling and coupled motion, and without kinetic complexities,   8 Klinman [72] has argued that, out of various thermodynamic and kinetic criteria, the most sensitive one for detection of tunneling is the breakdown of the above relationship, particularly for the secondary kinetic isotope effect [73]. In line with this, Cha et al. [74] have studied the oxidation of benzyl alcohol by NAD+, using six differently labeled alcohols as substrates:   in order to obtain all combinations of isotope effects. This reaction is suitable for exploring hydrogen tunneling because of the lack of any kinetic complexity, as it has a rate-limiting H-transfer step [75]. Cha et al. [74] have demonstrated that, in this reaction, the exponents in Eq. 8 are 3.58 and 10.2 for the primary and secondary kinetic isotope effect, respectively, indicating significant breakdown of the semi-classical upper limit. For hydrogen tunneling to occur, the reactive carbon atoms have been brought close together so that the classical energy barrier is penetrated. Thus, it appears that hydrogen tunneling is an additional general phenomenon which facilitates the YADH catalysis [23,76–78]. Leskovac et al. [79] have studied the primary kinetic isotope effects and the internal thermodynamics of the YADH-catalyzed oxidation of 2-propanol-h8 and 2-propanol-d8 with NAD+; the properties of this reaction were compared with non-enzymatic model redox reactions of N1-substituted-1,4(1H2)dihydronicotinamides and N1-substituted-1,4(1H2H)dihydronicotinamides with a number of various oxidizing agents. 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TI - The three zinc-containing alcohol dehydrogenases from baker's yeast, Saccharomyces cerevisiae
JF - FEMS Yeast Research
DO - 10.1111/j.1567-1364.2002.tb00116.x
DA - 2002-12-01
UR - https://www.deepdyve.com/lp/oxford-university-press/the-three-zinc-containing-alcohol-dehydrogenases-from-baker-s-yeast-CfZQBPDDW0
SP - 481
EP - 494
VL - 2
IS - 4
DP - DeepDyve
ER -