Using proton nuclear magnetic resonance to study the mode of ribonuclease
A inhibition by competitive and noncompetitive inhibitors
Kalyan Sundar Ghosh, Joy Debnath, Tanmaya Pathak, Swagata Dasgupta
*
Department of Chemistry, Indian Institute of Technology, Kharagpur, West Bengal 721302, India
article info
Article history:
Received 17 June 2008
Revised 2 September 2008
Accepted 4 September 2008
Available online 7 September 2008
Keywords:
Ribonuclease A
1
H NMR
Histidine pK
a
Competitive and noncompetitive inhibition
abstract
The C(2) proton resonances of the active site histidines (His 12 and His 119) of ribonuclease A have been
exploited to study the inhibition pattern of both noncompetitive (four green tea polyphenols and their
copper complexes) and competitive (3
0
-O-carboxy esters of thymidine and 3
0
-amino derivatives of uri-
dine) inhibitors. Competitive inhibitors devoid of any phosphate group have the ability to change the
pK
a
of the histidine residues at the active site. Their mode of inhibition, albeit competitive, is found to
be different compared to known phosphate inhibitors 2
0
-CMP and 3
0
-CMP as revealed by changes in
the pK
a
values. We find a correlation between the changes in the chemical shift of His 12 and the corre-
sponding inhibition constants (K
i
).
Ó 2008 Elsevier Ltd. All rights reserved.
The proton nuclear magnetic resonance (NMR) spectra of pro-
teins contain numerous overlapping resonances, which are not al-
ways appropriately resolved. However, the C(2) proton resonances
of histidine (His) residues can clearly be resolved for a few pro-
teins.
1
In case of bovine pancreatic ribonuclease A (RNase A), the
C(2)–H resonances of all four His residues are observed sepa-
rately.
1–3
Of these, His 12 and His 119 comprise the P
1
subsite of
the active site of the enzyme and are mainly involved in the phos-
phodiester hydrolysis mechanism.
4,5
In the most widely accepted
chemical mechanism,
6–8
His 12 acts as a base to deprotonate the
2
0
-oxygen atom of the substrate to form the cyclic 2
0
,3
0
-phosphate
intermediate. His 119 behaves as an acid to protonate the oxygen
atom of the leaving group. This mechanism was confirmed by site
directed mutagenesis studies.
8–10
Changes in proton resonances of the two active site His residues
due to changes in their protonation or deprotonation environment
can be correlated to the mode of RNase A inhibition. When a com-
petitive inhibitor binds to the enzyme, the chemical shifts (d)of
C(2)–H of the active site His residues are expected to change. How-
ever, for an inhibitor not going to the active site, little or no change
in the d value of C(2)–H is expected. This can easily be used for
classification of the type of inhibitor of RNase A. In this report
we have described the effect of binding of both competitive and
noncompetitive types of inhibitors on the chemical shifts of the
histidine residues of RNase A, particularly on the two histidines sit-
uated at the active site. To the best of our knowledge this is the first
time a report on the effect of any noncompetitive inhibitor on
RNase A histidines has been reported by NMR. The changes in
pK
a
of His 12 and His 119 on binding of the competitive inhibitors
were calculated from NMR titrations. Among competitive inhibi-
tors both phosphate and nonphosphate based inhibitors were em-
ployed. From the pK
a
change, different modes of inhibition by
phosphate and nonphosphate inhibitors were concluded. Finally
a correlation between the change in d of C(2)–H of His 12 on com-
plexation with competitive inhibitors and their reported inhibition
constants (K
i
) was performed. This correlation suggests that a mea-
sure in the change in d could be utilized to predict the K
i
of an
inhibitor. This study could also be extended to other proteins like
angiogenin,
11
eosinophil-derived neurotoxin (EDN),
12
etc., belong-
ing to the ribonuclease superfamily because the arrangement of
ribonucleolytic residues (two histidines and one lysine) is con-
served in all of them.
13
A rapid screening of inhibitors of such pro-
teins would allow the further development of potent inhibitors of
these proteins.
Exchangeable hydrogen atoms of RNase A were replaced with
deuterium by dissolving the protein in D
2
O with incubation for
20 min at 50 °C and then lyophilized following the procedure of
Quirk and Raines.
14
This procedure was performed thrice. The
lyophilized protein was dissolved in D
2
O containing 0.2 M NaCl.
The pH of the samples was measured with a microelectrode assem-
bled with a pH meter. Small amounts of DCl and NaOD were used
to adjust the pH
*
for the range 3–8, where pH
*
is the direct measure
of the pH which was not corrected for a deuterium isotope effect.
The [ligand] to [enzyme] molar ratio was maintained at 5:1 for
all the compounds.
1
H NMR spectra were recorded on a Bruker
400 MHz spectrometer at 22.5 °C. The acquisition time was 2 s
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.014
* Corresponding author. Tel.: +91 3222 283306; fax: +91 3222 255303.
E-mail addresses: swagata@chem.iitkgp.ernet.in (S. Dasgupta), swagata_dasgup-
ta@hotmail.com (S. Dasgupta).
Bioorganic & Medicinal Chemistry Letters 18 (2008) 5503–5506
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl