THERMOKINETIC STUDIES OF THE IRREVERSIBLE INHIBITION OF SINGLE-SUBSTRATE, ENZYME-CATALYZED REACTIONS

A thermokinetic ratio method for the irreversible inhibition of single -substrate enzyme-catalyzed reactions is proposed in this paper, By an alyzing a measured curve this method can be used to calculate the appa rent rate constant of the inhibited reaction without letting the react ion go to completion. Using the LKB-2107 batch microcalorimeter, the a rginase-catalyzed hydrolysis of L-arginine in the presence of p-chloro mercuribenzoate (PCMB) has been studied and PCMB established as an irr eversible mixed inhibitor, The second-order rate constants for inhibit ion 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(-1) s(-1) a nd K-ESI = 35.2 M(-1) s(-1), respectively, at 298.15 K, Chemical modif ication with PCMB indicates that arginase contains three reactive cyst einyl residues at most but these residues are not present at the activ e site of arginase.