13C and1H NMR Studies of Ionizations and Hydrogen Bonding in Chymotrypsin-Glyoxal Inhibitor Complexes

Edward Spink, Sonya Cosgrove, Louis Rogers, Chandralal Hewage, J. Paul G. Malthouse
2007 Journal of Biological Chemistry  
Benzyloxycarbonyl (Z)-Ala-Pro-Phe-glyoxal and Z-Ala-Ala-Phe-glyoxal have both been shown to be inhibitors of ␣-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 pK a values of ϳ4.0 and ϳ10.5, 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. 13 C NMR has been
more » ... ed to show that at low pH the glyoxal keto carbon is sp 3 -hybridized with a chemical shift of ϳ100.7 ppm and that the aldehyde carbon is hydrated with a chemical shift of ϳ91.6 ppm. The signal at ϳ100.7 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 pK a of ϳ4.5, the aldehyde carbon dehydrates to give a signal at ϳ205.5 ppm and the hemiketal forms an oxyanion at ϳ107.0 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 (pK a ϳ9.2). On binding either Z-Ala-Pro-Phe-glyoxal or Z-Ala-Ala-Phe-glyoxal to ␣-chymotrypsin at 4 and 25°C, 1 H NMR is used to show that the binding of these glyoxal inhibitors raises the pK a 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 pK a 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 pK a value of the oxyanion forming a zwitterionic tetrahedral intermediate during catalysis. Specific substrate-derived glyoxal inhibitors have been shown to be potent inhibitors of the serine proteinases (1-4). Z 4 -Ala-Pro-Phe-glyoxal is an extremely potent reversible inhibitor of ␦-chymotrypsin with an apparent disassociation constant of 25 Ϯ 8 nM at pH 7.0 (1).
doi:10.1074/jbc.m611394200 pmid:17213185 fatcat:vokoar2gu5gy7b6e5wlvlk6w6i