The role of methionine-192 of the chymotrypsin active site in the binding and catalysis of mono(amino acid) and peptide substrates

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Abstract
Specific oxidation of the Met-192 residue in [bovine] .delta.-chymotrypsin to methionine sulfoxide results in a 2-fold increase in Km(app) and unchanged kcat in the hydrolysis of N-acetyl mono(amino acid) amide substrates. The catalyzed hydrolyses of N-acetyl dipeptide amide substrates by (methionine sulfoxide)-192-.delta.-chymotrypsin (Ms-.delta.-Cht) shows a 4- to 5-fold decrease in kcat and unchanged Km(app) with respect to .delta.-chymotrypsin. Hydrolysis of .alpha.-casein by MS-.delta.-Cht shows a similar 4.2-fold decrease in kcat. These results imply that the Met-192 acts differently with substrates that bind only in the primary, S1, binding site (i.e., AcPheNH2) from those that bind to more extended regions of the enzyme active site. In the binding of AcPheNH2 and AcTrpNH2, the results support a mechanism in which the Met-192 acts to slow the rate of sustrate dissociation from the Michaelis complex to free substrate and enzyme. This is in agreement with the x-ray crystallographic structure of dioxane inhibited .alpha.-chymotrypsin. This mechanism is not apparent when peptide and protein substrates bind. The .apprx. 5-fold decrease in kcat of the hydrolysis of polypeptide substrates on Met-192 modification shows a small, but significant, catalytic contribution of the Met-192 toward the lowering of the activation energy for polypeptide substrate hydrolysis by chymotrypsin. This may support the crystallographic model of Fersht et al. which proposed that the Met-192 participates in the distortion of bound polypeptide substrates toward the reaction transition-state configuration and thus, plays a role in catalysis. If this mechanism does occur, the effect is small, only contributing .apprx. 1 kcal/mol to the lowering of the reaction activation energy.