Mechanism of the reaction of papain with substrate-derived diazomethyl ketones. Implications for the difference in site specificity of halomethyl ketones for serine proteinases and cysteine proteinases and for stereoelectronic requirements in the papain catalytic mechanism

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Abstract
The reactions of papain (EC 3.4.22.2) with substrate-derived diazomethyl ketones reported by Leary et al. are unusual in that these reagents fail to react with low-molecular weight thiols and the rate of reaction with the papain thiol group does not decrease to near-zero values across a pKa of 4 as the pH is decreased. Existing data suggest an interpretation involving neighboring-group participation via transient thiohemiketal formation, rate-determining protonation by imidazolium ion and alkylation on sulfur via a 3-membered cyclic transition state. Implications for the difference in site-specificity exhibited by halomethyl ketones in their reactions with serine proteinases and cysteine proteinases and stereoelectronic requirements in the mechanism of papain-catalyzed hydrolysis are discussed. The possibility of 2 tetrahedral intermediates between adsorptive complex and acyl-enzyme is indicated.