The kinetic mechanism of beef kidney D-aspartate oxidase

A. Negri, V. Massey, C. H. Williams, L. M. Schopfer

Research output: Contribution to journalArticlepeer-review

21 Scopus citations

Abstract

The mechanism of action of the flavoprotein D-aspartate oxidase (EC 1.4.3.1) has been investigated by steady-state and stopped flow kinetic studies using D-aspartate and O2 as substrates in 50 mM KP(i), 0.3 mM EDTA, pH 7.4, 4°C. Steady-state results indicate that a ternary complex containing enzyme, O2, and substrate (or product) is an obligatory intermediate in catalysis. The kinetic parameters are turnover number = 11.1 s-1, K(m(D-Asp)) = 2.2 x 10-3 M, K(m(O2)) = 1.7 x 10-4 M. Rapid reaction studies show that 1) the reductive half reaction is essentially irreversible with a maximum rate of reduction of 180 s-1; 2) the free reduced enzyme cannot be the species which is reoxidized during turnover since its reoxidation by oxygen (second order rate constant equal to 5.3 x 102 M-1 S-1) is too slow to be of relevance in catalysis; 3) reduced enzyme can bind a lignad rapidly and be reoxidized as a complex at a rate faster than that observed for the free reduced enzyme; 4) the rate of reoxidation of reduced enzyme by oxygen during turnover is dependent on both O2 and D-aspartate concentrations (second order rate constant of reaction between O2 and reduced enzyme-substrate complex equal to 6.2 x 104 M-1 s-1); and 5) the rate-limiting step in catalysis occurs after reoxidation of the enzyme and before its reduction in the following turnover. A mechanism involving reduction of enzyme by substrate, dissociation of product from reduced enzyme, binding of a second molecule of substrate to the reduced enzyme, and reoxidation of the reduced enzyme-substrate complex is proposed for the enzyme-catalyzed oxidation of D-aspartate.

Original languageEnglish (US)
Pages (from-to)13557-13563
Number of pages7
JournalJournal of Biological Chemistry
Volume263
Issue number27
StatePublished - 1988

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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