13C-NMR study of the inhibition of δ-chymotrypsin by a tripeptide-glyoxal inhibitor

Abstract

A new inhibitor, Z-Ala-Pro-Phe-glyoxal (where Z is benzyloxycarbonyl),has been synthesized and shown to be a competitive inhibitor of δ-chymotrypsin, with a Ki of 25±8nM at pH7.0 and 25°C. Z-Ala-Pro-[1-13C]Phe-glyoxal and Z-Ala-Pro-[2-13C]Phe-glyoxal have been synthesized, and 13C-NMR has been used to determine how they interact with δ-chymotrypsin. Using Z-Ala-Pro-[2-13C]Phe-glyoxal we have detected a signal at 100.7p.p.m. which we assign to the tetrahedral adduct formed between the hydroxy group of Ser-195 and the 13C-enriched keto-carbon of the inhibitor. This signal is in a pH-dependent slow exchange with a signal at 107.6p.p.m. which depends on a pKa of ∼ 4.5, which we assign to oxyanion formation. Thus we are the first to detect an oxyanion pKa in a reversible chymotrypsin—inhibitor complex. A smaller titration shift of 100.7p.p.m. to 103.9p.p.m. with a pKa of ∼ 5.3 is also detected due to a rapid exchange process. This pKa is also detected with the Z-Ala-Pro-[1-13C]Phe-glyoxal inhibitor and gives a larger titration shift of 91.4p.p.m. to 97.3p.p.m., which we assign to the ionization of the hydrated aldehyde hydroxy groups of the enzyme-bound inhibitor. Protonation of the oxyanion in the oxyanion hole decreases the binding efficiency of the inhibitor. From this decrease in binding efficiency we estimate that oxyanion binding in the oxyanion hole reduces the oxyanion pKa by 1.3 pKa units. We calculate that the pKas of the oxyanions of the hemiketal and hydrated aldehyde moieties of the glyoxal inhibitor are both lowered by 6.4–6.9 pKa units on binding to chymotrypsin. Therefore we conclude that oxyanion binding in the oxyanion hole has only a minor role in decreasing the oxyanion pKa. We also investigate how the inhibitor breaks down at alkaline pH, and how it breaks down at neutral pH in the presence of chymotrypsin.