Pyridoxamine-pyruvate transaminase. 2. Temperature-jump and stopped-flow kinetic investigation of the rates and mechanism of the reaction of 5'-deoxypyridoxal with the enzyme.

Abstract

The kinetics and mechanism of enzymatic Schiff base formation and hydrolysis were investigated by rapid re- actions measurements of 5'-deoxypyridoxal with pyridox- amine-pyruvate transaminase (EC 2.6.1.30). The dissociation rate constant, koff, was determined as a function of pH over the range pH 7-9 by a stopped-flow method in which the nascent free enzyme was trapped by the potent bisubstrate analogue inhibitor, N-pyridoxyl-L-alanine. The values of kofr increase with pH and are dependent upon a pK, (app) of 8.35 which is assigned to the pyridine nitrogen of the Schiff base formed between 5'-deoxypyridoxal and an t-amino group of the active site lysine. The rate-determining step in the disso- ciation reaction is assigned to the separation of the components The wealth of spectral detail accompanying transformations between the various intermediates involved in pyridoxal phosphate dependent enzyme catalyzed transamination has stimulated important efforts to elucidate the mechanistic de- tails of this reaction by the temperature-jump technique. The prototypic enzyme for these investigations has been aspartate