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

The aldehyde inhibitor Z-Ala-Ala-Phe-CHO has been synthesized and shown by (13)C-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 pK(a) value of ~ 7 showing that chymotrypsin reduces the oxyanion pK(a) values by ~ 5.6 pK(a) 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 (1)H-NMR we have shown that at both 4 and 25 °C the histidine pK(a) 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 pK(a) and that the aldehyde hydrogen must be replaced by a larger group to raise the histidine pK(a) > 10 and give stereospecific formation of tetrahedral intermediates. The results show that a large increase in the pK(a) of the active site histidine is not needed for the active site serine hydroxyl group to have an effective molarity of 6000.