The kinetic mechanism of beef kidney D-aspartate oxidase.
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 KPi, 0.3 mM EDTA, pH 7.4, 4 degrees 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, Km(D-Asp) = 2.2 x 10(-3) M, Km(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 10(2) M-1 s-1) is too slow to be of relevance in catalysis; 3) reduced enzyme can bind a ligand 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 10(4) 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.
D000594:Amino Acid Oxidoreductases / Q000378:metabolism*; D000818:Animals; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D002384:Catalysis; D002417:Cattle; D003605:D-Amino-Acid Oxidase / Q000378:metabolism; D050804:D-Aspartate Oxidase; D006861:Hydrogen Peroxide / Q000378:metabolism; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D010071:Oxaloacetates / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D013053:Spectrophotometry
D010071:Oxaloacetates; D001224:Aspartic Acid; C031948:iminoaspartic acid; D006861:Hydrogen Peroxide; D000594:Amino Acid Oxidoreductases; D050804:D-Aspartate Oxidase; D003605:D-Amino-Acid Oxidase; D010100:Oxygen