Hemiacetal stabilization in a chymotrypsin inhibitor complex and the reactivity of the hydroxyl group of the catalytic serine residue of chymotrypsin

Files in This Item:
File Description SizeFormat 
Accepted-Rev-manuscript-v3plusFigsetcCORRECTED.pdf3.47 MBAdobe PDFDownload
Title: Hemiacetal stabilization in a chymotrypsin inhibitor complex and the reactivity of the hydroxyl group of the catalytic serine residue of chymotrypsin
Authors: Cleary, Jennifer A.
Doherty, William
Evans, Paul
Malthouse, J.Paul G.
Permanent link: http://hdl.handle.net/10197/5629
Date: Jun-2014
Abstract: The aldehyde inhibitor Z-Ala-Ala-Phe-CHO has been synthesized and shown by 13C-NMR to react with the active site serine hydroxyl group of alpha-chymotrypsin to form two diastereomeric hemiacetals. For both hemiacetals oxyanion formation occurs with a pKa value of ~ 7 showing that chymotrypsin reduces the oxyanion pKa values by ~ 5.6 pKa units and stabilizes the oxyanions of both diastereoisomers by ~ 32 kJ mol− 1. As pH has only a small effect on binding we conclude that oxyanion formation does not have a significant effect on binding the aldehyde inhibitor. By comparing the binding of Z-Ala-Ala-Phe-CHO with that of Z-Ala-Ala-Phe-H we estimate that the aldehyde group increases binding ~ 100 fold. At pH 7.2 the effective molarity of the active site serine hydroxy group is ~ 6000 which is ~ 7 × less effective than with the corresponding glyoxal inhibitor. Using 1H-NMR we have shown that at both 4 and 25 °C the histidine pKa is ~ 7.3 in free chymotrypsin and it is raised to ~ 8 when Z-Ala-Ala-Phe-CHO is bound. We conclude that oxyanion formation only has a minor role in raising the histidine pKa and that the aldehyde hydrogen must be replaced by a larger group to raise the histidine pKa > 10 and give stereospecific formation of tetrahedral intermediates. The results show that a large increase in the pKa of the active site histidine is not needed for the active site serine hydroxyl group to have an effective molarity of 6000.
Funding Details: Irish Research Council
Science Foundation Ireland
University College Dublin
Type of material: Journal Article
Publisher: Elsevier
Copyright (published version): 2014 Elsevier
Keywords: Chymotrypsin;Aldehyde inhibitor;Hemiacetal;Oxyanion;Effective molarity
DOI: 10.1016/J.BBAPAP.2014.03.008
Language: en
Status of Item: Peer reviewed
Appears in Collections:Biomolecular and Biomedical Science Research Collection

Show full item record

Citations 50

Last Week
Last month
checked on Jun 15, 2018

Google ScholarTM



This item is available under the Attribution-NonCommercial-NoDerivs 3.0 Ireland. No item may be reproduced for commercial purposes. For other possible restrictions on use please refer to the publisher's URL where this is made available, or to notes contained in the item itself. Other terms may apply.