13C and1H NMR studies of ionizations and hydrogen bonding in chymotrypsin-glyoxal inhibitor complexes

Abstract

Benzyloxycarbonyl (Z)-Ala-Pro-Phe-glyoxal and Z-Ala-Ala-Phe-glyoxal have both been shown to be inhibitors of α-chymotrypsin with minimal K i values of 19 and 344 nM, respectively, at neutral pH. These K i values increased at low and high pH with pKa values of ∼4.0 and ∼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 ∼100.7 ppm and that the aldehyde carbon is hydrated with a chemical shift of ∼91.6 ppm. The signal at ∼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 ∼4.5, the aldehyde carbon dehydrates to give a signal at ∼205.5 ppm and the hemiketal forms an oxyanion at ∼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 ∼9.2). On binding either Z-Ala-Pro-Phe-glyoxal or Z-Ala-Ala-Phe-glyoxal to α-chymotrypsin at 4 and 25°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°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. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.