Thermokinetic studies of the irreversible inhibition of single-substrate, enzyme-catalyzed reactions

Abstract

A thermokinetic ratio method for the irreversible inhibition of single-substrate enzymecatalyzed reactions is proposed in this paper. By analyzing a measured curve this method can be used to calculate the apparent rate constant of the inhibited reaction without letting the reaction go to completion. Using the LKB-2107 batch microcalorimeter, the arginase-catalyzed hydrolysis of L-arginine in the presence of p-chloromercuribenzoate (PCMB) has been studied and PCMB established as an irreversible mixed inhibitor. The second-order rate constants for inhibition of arginase by PCMB in the absence and presence of L-arginine have been determined by this ratio method to be k EI = 94.4 M − s −1 and k ESI = 35.2 M −1 s −1, respectively, at 298.15 K. Chemical modification with PCMB indicates that arginase contains three reactive cysteinyl residues at most but these residues are not present at the active site of arginase.