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

•Z-Ala-Ala-Phe-CHO forms two diastereomeric hemiacetals with chymotrypsin.•The oxyanions of both hemiacetals have pKa values of ~ 7.•The oxyanion has only a small effect on inhibitor binding and on the pKa of his-57.•The aldehyde group of the inhibitor increases inhibitor binding 100-fold.•The effective molarity of the active site serine hydroxyl is ~ 6000 M.

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.