Comparison of the kinetics of the papain-catalyzed hydrolysis of glycine- and alanine-based esters and thiono esters

The kinetic constants for the papain-catalyzed hydrolysis of a series of substrates with glycine or alanine in the P1 position are discussed. The substrates have N-benzoyl, N-(p-nitrobenzoyl), N-(beta-phenylpropionyl), or N-(methyloxycarbonyl)phenylalanine attached to the P1 moiety, and kinetic constants are obtained for both esters and thiono esters. The results for the hydrolysis of esters can be readily interpreted in terms of the known specificity of papain. For any glycine ester the change in kcat/Km upon substituting C=S for C=O or upon substituting an alpha-CH3 group is minimal. However, upon making both these substitutions, i.e., going from a glycine ester to an alanine thiono ester substrate, larger changes are seen for this ratio. Data for N-benzoyl- and N-(beta-phenylpropionyl)glycine and -alanine methyl thiono esters show that k2 is the parameter most affected by the double C=S and alpha-CH3 substitution. A further conclusion is that the deacylation rate constants for any pair of glycine and alanine dithioacyl papains are similar; e.g., for the intermediates based on the "good" substrates PheAla and PheGly k3 differs by only 20%. This is a surprising finding in light of the very different conformations and interactions of the bound acyl groups revealed by resonance Raman spectroscopy and raises the possibility that specific stereochemical effects, such as the oxyanion hole and general base catalysis, are not operating in the hydrolysis of dithioacyl papains.