Insight into the catalysis of hydrolysis of four newly synthesized substrates by papain: A proton inventory study

We synthesized the following four new peptide substrates, Suc-Phe-Leu-pNA, Suc-Phe-Leu-NMec, Suc-Phe-Leu-ONPh, and Pht-Phe-Leu-pNA, and we applied the proton inventory method to their hydrolysis by papain. Useful relationship s between the rate constants of the catalytic reaction have been establishe d and contributed to the elucidation of the hydrolytic mechanism of papain. For all amide substrates, the parameter K-s and the rate constants k(1), k (-1), and k(2) were estimated. Moreover, it was found that k(cat)/K-m = k(1 ) for all four substrates, while two exchangeable hydrogenic sites, one in the ground state and another in the transition state, generate an inverse i sotope effect during the reaction governed by this parameter. The proton in ventories of both k(2) and k(3) are essentially linear, whatever the acyl m oiety and/or the leaving group of the substrate. The proton inventories of K-s are also essentially linear for all amide substrates, while the observe d large isotope effect of about 3 to 9 originates from a single hydrogenic site in the product state. This latter, in agreement to both the small tran sition state fractionation factors found for k(cat)/K-m (or k(1)) and the u nit ground-slate fractionation factors found for k(2), argues for the forma tion of a tetrahedral adduct during the reaction governed by the k(1) param eter. Furthermore, papain acts as a one-proton catalyst during acylation or deacylation, both of which proceed through similar concerted reaction path ways, where a nucleophilic attack is accompanied by the movement of one pro ton.