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THE ACYL-ENZYME INTERMEDIATE AND THE KINETIC MECHANISM OF THE GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE REACTION.

C. Furfine,S. Velick

Published 1965 in Journal of Biological Chemistry

ABSTRACT

The acyl-enzyme compound formed by n-glyceraldehyde 3-phosphate dehydrogenase has been studied by several workers in a variety of ways (l), primarily in experiments at high enzyme concentrations and in many instances with model substrates. In the present investigation an attempt is made to apply kinetic criteria to the mechanism of the reversible oxidative phosphorylation of the natural substrates. Although the oxidative and acyl transfer steps are clearly separable at substrate level enzyme concentrations, the initial velocity kinetics at low enzyme concentration is described by a random order of substrate addition to enzyme with the formation of a kinetically important quaternary enzyme-substrate complex. One does not obtain, in initial velocity measurements, the same kinetic signs of group transfer through an enzyme-bound intermediate that are observed, for example, with the glutamate-oxaloacetate transaminase (2). It is found that the rate-limiting step in glyceraldehyde 3-phosphate oxidation at pH 7.4 is the acyl group transfer from a site on the enzyme to an external acceptor. The properties of the partial reactions are such that in the physiological pH region the kinetics is dominated by a high steady state concentration of the intermediate. There is a large effect of the intermediate on the atypical interactions of pyridine nucleotide with enzyme, apparently mediated by protein isomerizations. Events which might not be recognized in a purely kinetic approach are clarified by an examination of isolated enzyme-substrate interactions and of the equilibria of the partial reactions in which the enzyme is a stoichiometric participant.

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