The kinetic mechanism of D-amino acid oxidase with D-alpha-aminobutyrate as substrate. Effect of enzyme concentration on the kinetics.

P. F. Fitzpatrick, V. Massey

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Abstract

The kinetic mechanism of hog kidney D-amino acid oxidase with D-alpha-aminobutyrate as substrate has been examined in detail using a combination of steady state and rapid reaction methods. At concentrations of D-alpha-aminobutyrate below 0.5 mM, the rapid reaction and steady state results are consistent with the mechanism previously proposed for D-alanine (Massey, V., and Gibson, Q. H. (1964) Fed. Proc. 23, 18-29; Porter, D. J. T., Voet, J. G., and Bright, H. J. (1977) J. Biol. Chem. 252, 4464-4473). Both flavin reduction by D-alpha-aminobutyrate and reoxidation are quite rapid. Release of product from the oxidized enzyme has been measured directly and matches the turnover number at infinite concentrations of both substrates. Substitution of deuterium for the alpha-hydrogen decreases the rate of reduction 1.4-fold, without any effect on the apparent Kd. Computer simulations show that the kinetic isotope effects on the reductive half-reaction with D-alanine reported by Porter et al. (see above reference) can be explained using a two-step model with a kinetic isotope effect of 1.75 on the limiting rate of reduction. The effect of enzyme concentration on the kinetics has been examined in some detail. With D-alanine as substrate, increasing the enzyme concentration over the range 29 nM to 17 microM resulted in less than a 2-fold decrease in the turnover number. The Kd for benzoate binding also decreased marginally with increasing enzyme concentration. The effect of enzyme concentration is consistent with a decrease in the rate of release of ligands from the oxidized enzyme as the enzyme concentration is increased.

Original languageEnglish (US)
Pages (from-to)12916-12923
Number of pages8
JournalJournal of Biological Chemistry
Volume257
Issue number21
StatePublished - Nov 10 1982

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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