Kinetics of the Interaction between Pig-Plasma Benzylamine Oxidase and Hydrazine Derivatives

Abstract

1 The interaction between hydrazines and pyridoxal phosphate in pig plasma benzylamine oxidase has been studied by stopped-flow techniques. The rapid formation of chromophoric enzyme-inhibitor complexes follows second-order kinetics and rate constants for a number of hydrazine derivatives are reported. Reaction rates with enzyme-bound pyridoxal phosphate are 20–1000 times higher than with free pyridoxal phosphate. 2 Analysis of the pH-dependence of second-order rate constants for the formation of enzyme-inhibitor complexes shows that the protonated form of the hydrazines is non-reactive, and that the reaction is dependent upon protonation of an ionizing group in the enzyme with a pKa close to 9. 3 The second-order reaction between enzyme and phenylhydrazine is competitive with the second-order reduction of the enzyme by substrate, supporting the idea that Schiff-base formation between substrate and enzyme-bound pyridoxal phosphate is an obligatory step in the catalytic mechanism. Hence it follows that the formation of enzyme-inhibitor complexes may be considered as a model for the interaction between substrates and the prosthetic group of the enzyme. 4 The interaction between enzyme and phenylhydrazine does not result in any pronounced electron paramagnetic spectral changes, indicating that the spectral changes observed on reduction of the enzyme by substrate cannot be ascribed to the formation of an enzyme-substrate complex.