Z-Ala-Pro-OH

Benzyloxycarbonyl (Z)-Ala-Pro-Phe-glyoxal and Z-Ala-Ala-Phe-glyoxal have both been shown to be inhibitors of alpha-chymotrypsin with minimal Ki values of 19 and 344 nM, respectively, at neutral pH. These Ki values increased at low and high pH with pKa values of approximately 4.0 and approximately 10.5, respectively. By using surface plasmon resonance, we show that the apparent association rate constant for Z-Ala-Pro-Phe-glyoxal is much lower than the value expected for a diffusion-controlled reaction. 13C NMR has been used to show that at low pH the glyoxal keto carbon is sp3-hybridized with a chemical shift of approximately 100.7 ppm and that the aldehyde carbon is hydrated with a chemical shift of approximately 91.6 ppm. The signal at approximately 100.7 ppm is assigned to the hemiketal formed between the hydroxy group of serine 195 and the keto carbon of the glyoxal. In a slow exchange process controlled by a pKa of approximately 4.5, the aldehyde carbon dehydrates to give a signal at approximately 205.5 ppm and the hemiketal forms an oxyanion at approximately 107.0 ppm. At higher pH, the re-hydration of the glyoxal aldehyde carbon leads to the signal at 107 ppm being replaced by a signal at 104 ppm (pKa approximately 9.2). On binding either Z-Ala-Pro-Phe-glyoxal or Z-Ala-Ala-Phe-glyoxal to alpha-chymotrypsin at 4 and 25 degrees C, 1H NMR is used to show that the binding of these glyoxal inhibitors raises the pKa value of the imidazolium ion of histidine 57 to a value of >11 at both 4 and 25 degrees C. We discuss the mechanistic significance of these results, and we propose that it is ligand binding that raises the pKa value of the imidazolium ring of histidine 57 allowing it to enhance the nucleophilicity of the hydroxy group of the active site serine 195 and lower the pKa value of the oxyanion forming a zwitterionic tetrahedral intermediate during catalysis.

Two new inhibitors in which the terminal α-carboxyl groups of Z-Ala-Ala-Phe-COOH and Z-Ala-Pro-Phe-COOH have been replaced with a proton to give Z-Ala-Ala-Phe-H and Z-Ala-Pro-Phe-H, respectively, have been synthesized. Using these inhibitors, we estimate that for α-chymotrypsin and subtilisin Carlsberg the terminal carboxylate group decreases the level of inhibitor binding 3-4-fold while a glyoxal group increases the level of binding by 500-2000-fold. We show that at pH 7.2 the effective molarities of the catalytic hydroxyl group of the active site serine are 41000-229000 and 101000-159000 for α-chymotrypsin and subtilisin Carlsberg, respectively. It is estimated that oxyanion stabilization and the increased effective molarity of the catalytic serine hydroxyl group can account for the catalytic efficiency of the reaction. We argue that substrate binding induces the formation of a strong hydrogen bond or low-barrier hydrogen bond between histidine-57 and aspartate-102 that increases the pK(a) of the active site histidine, allowing it to be an effective general base catalyst for the formation of the tetrahedral intermediate and increasing the effective molarity of the catalytic hydroxyl group of serine-195. A catalytic mechanism for acyl intermediate formation in the serine proteases is proposed.

A new inhibitor, Z-Ala-Pro-Phe-glyoxal (where Z is benzyloxycarbonyl),has been synthesized and shown to be a competitive inhibitor of delta-chymotrypsin, with a K(i) of 25+/-8 nM at pH 7.0 and 25 degrees C. Z-Ala-Pro-[1-(13)C]Phe-glyoxal and Z-Ala-Pro-[2-(13)C]Phe-glyoxal have been synthesized, and (13)C-NMR has been used to determine how they interact with delta-chymotrypsin. Using Z-Ala-Pro-[2-(13)C]Phe-glyoxal we have detected a signal at 100.7 p.p.m. which we assign to the tetrahedral adduct formed between the hydroxy group of Ser-195 and the (13)C-enriched keto-carbon of the inhibitor. This signal is in a pH-dependent slow exchange with a signal at 107.6 p.p.m. which depends on a pK(a) of approximately 4.5, which we assign to oxyanion formation. Thus we are the first to detect an oxyanion pK(a) in a reversible chymotrypsin-inhibitor complex. A smaller titration shift of 100.7 p.p.m. to 103.9 p.p.m. with a pK(a) of approximately 5.3 is also detected due to a rapid exchange process. This pK(a) is also detected with the Z-Ala-Pro-[1-(13)C]Phe-glyoxal inhibitor and gives a larger titration shift of 91.4 p.p.m. to 97.3 p.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 pK(a) by 1.3 pK(a) units. We calculate that the pK(a)s of the oxyanions of the hemiketal and hydrated aldehyde moieties of the glyoxal inhibitor are both lowered by 6.4-6.9 pK(a) units on binding to chymotrypsin. Therefore we conclude that oxyanion binding in the oxyanion hole has only a minor role in decreasing the oxyanion pK(a). We also investigate how the inhibitor breaks down at alkaline pH, and how it breaks down at neutral pH in the presence of chymotrypsin.