Kinetics of inactivation of aminoacylase by 2-chloromercuri-4-nitrophenol: a new type of complexing inhibitor.

Abstract

The kinetic theory of the substrate reaction during modification of enzyme activity previously described [Tsou (1988) Adv. Enzymol. 61, 381-436] has been applied to a study of the inactivation kinetics of aminoacylase by 2-chloromercuri-4-nitrophenol (MNP). The results indicate that the mechanism of reaction between MNP and aminoacylase is a special type of irreversible inhibition. The main features of this type of inhibitor are as follows: (i) the reaction kinetics of inhibitor with enzyme is a single exponential process; (ii) inhibition shows a noncompetitive, complexing behavior; (iii) the first inhibitor-enzyme complex, EI, still has some enzyme activity, and hence the plot of [P]infinity versus the reciprocal of inhibitor concentration gives a straight line with a positive intercept at the ordinate. On the basis of the kinetic equation of substrate reaction in the presence of the inhibitor, a plotting method has been developed for determining the inhibition kinetic constants. As an example, all reaction kinetic constants of aminoacylase with 2-chloromercuri-4-nitrophenol have been determined. The results of the present study suggest that the essential thiol group at the active site of aminoacylase may have a significant effect on the catalytic step but is not involved in substrate binding.