PH DEPENDENCIES OF INDIVIDUAL RATE CONSTANTS IN PAPAIN-CATALYZED REACTIONS

This report describes measurements of acylation rate constants (k2) and apparent binding constants (Ks = (k-1 + k2)/k1) for substrates of papain over a range of pH. The Michaelis-Menten parameters (km and kcat) for the papaincatalyzed hydrolysis of isopropyl and methyl hippurates and isopropyl, benzyl, and p-methoxyphenyl N-methanesulfonylglycinates have been determined over a range of pH at 35°. Assuming a three-step mechanism: [formula omitted] k2 and Ks may be calculated for the isopropyl esters because kcat for these substrates lies well below k3 for the common acyl-enzyme. This calculation requires a bona fide value for k3 (supplied by kcat for the other esters where deacylation is shown to be rate determining) and the assumptions that the rate-determining step is acylation for the isopropyl esters and that the three-step mechanism is valid for all substrates. These assumptions are more directly validated by experiment than those used in previous attempts to dissect composite Michaelis-Menten parameters. The pH dependence of k2 is found to be bell shaped with pK1 ~ 4.5 and pK2 ~ 8.5. Ks is probably a true binding constant (k-1 ≫ k2) since it is found to be invariant in the pH range where k2 varies significantly, and this conclusion is strengthened by the observation that uncharged reversible inhibitors with substrate-like structures have pH-independent competitive inhibition constants. A new mechanism is proposed for papain-catalyzed hydrolyses where an imidazole gives rise to the acid pKa in acylation and deacylation by acting as a general base abstracting a proton from thiol and nucleophile, respectively; the abnormally low pKa is caused by hydrogen bonding with an asparagine. The base pKa in acylation is due to the ionization of a thiol which reacts in its un-ionized form. © 1969, American Chemical Society. All rights reserved.