Cleary, Jennifer A.Jennifer A.ClearyDoherty, WilliamWilliamDohertyEvans, PaulPaulEvansMalthouse, J.Paul G.J.Paul G.Malthouse2014-06-042014-06-042014 Elsev2014-06Biochimica Et Biophysica Acta (BBA) -Proteins and Proteomicshttp://hdl.handle.net/10197/5629The 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.enThis is the author’s version of a work that was accepted for publication in Biochimica Et Biophysica Acta (BBA) -Proteins and Proteomics. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Biochimica Et Biophysica Acta (BBA) -Proteins and Proteomics (VOL1844, ISSUE 6, (2014)) DOI:10.1016/J.BBAPAP.2014.03.008ChymotrypsinAldehyde inhibitorHemiacetalOxyanionEffective molarityJournal Article184461119112710.1016/J.BBAPAP.2014.03.0082014-04-16https://creativecommons.org/licenses/by-nc-nd/3.0/ie/