PAPAIN-CATALYSED HYDROLYSIS OF SOME HIPPURIC ESTERS. A NEW MECHANISM FOR PAPAIN-CATALYSED HYDROLYSIS

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Abstract
The Michaelis-Menten parameters for the papain-catalysed hydrolysis of a number of alkyl, aryl and alkyl-thiol esters of hippuric acid have been determined. For all the aryl esters and most of the alkyl esters studied, the catalytic constant, kg, is 2-3 sec. -1 and most probably represents deacylation of the common intermediate, hippuryl-papain. Two alkyl esters and hippurylamide, however, have catalytic rate constants, k0, less than 2-3 sec.-1. It is possible to interpret all the available kinetic data in terms of a three-step mechanism in which an enzyme-substrate complex is first formed, followed by acylation of the enzyme through an essential thiol group, followed by deacylation of the acyl-enzyme. The logarithm of the ratio of the Michaelis-Menten parameters, which reflect the acylation rate constant, for four aryl esters of hippuric acid studied give a linear Hammett plot against the substituent constant, [delta]. Arguments are presented that indicate acid as well as neucleophilic catalysis in the acylation process and that the most likely proton donor is an imidazolium ion. It is suggested that this imidazolium ion is part of the same histidine residue that has been tentatively implicated in the deacylation process (Lowe & Williams, 1965b). A new mechanism is proposed for the papain-catalysed hydrolysis of N-acyl- [alpha]-amino acid derivatives.