Studies on the Mechanism of D-Amino Acid Oxidase.

Several aspects of the mechanism of D-amino acid oxidase have been studied. Cysteamine and glyoxylate form thiazolidine-2-carboxylic acid, a substrate for D-amino acid oxidase, as Hamilton et al. proposed (Hamilton, G. S., Buckthal, D. J., Mortensen, R. M., and Zerby, K. W. (1979) Proc. Nat. Acad. USA 76, 2625-2629), with a second order rate constant of 84 M('-1) min('-1) at pH 7.5, 37(DEGREES)C. The product of the enzymic oxidation of thiazolidine-2-carboxylic acid is (DELTA)(,2)-thiazoline-2-carboxylic acid. Both the steady state and the reductive half reaction kinetics are similar to those with D-proline. D-amino acid oxidase has apparent activity with L-thiazolidine-2-carboxylic acid due to its rapid isomerization to the D-isomer. The results of steady state and rapid reaction experiments with D-(alpha)-aminobutyric acid as substrate fit a mechanism in which flavin reduction and reoxidation are both very rapid; the rate-determining step is product release from oxidized enzyme. The steady state kinetics exhibit negative cooperativity at concentrations of D-(alpha)-aminobutyric acid above 0.5 mM. Use of D-(alpha)-{('2)H}-aminobutyric acid decreases the limiting rate of reduction by a factor of 1.4. The turnover number, (phi)(,0)('-1), decreases severalfold as the enzyme concentration is increased from 10('-8) to 10('-5) M; (phi)(,A) and ('(phi))O(,2) are unaffected. There is only a small effect on benzoate binding. Two protons are released to solution during the reductive half reaction. The first is released during reduction and the second upon dissociation of the reduced enzyme-imino acid charge-transfer complex. Modification of the active site lysine, histidine, or tyrosine has no effect on the ability of the enzyme to stabilize the benzoquinonoid form of 8-mercapto-FAD. Modification of the active site arginine results in loss of the flavin. 2-Thio-FAD binds D-amino acid oxidase with a K(,d) of 6 (mu)M. The pK of 2-thio-FAD is lowered to 8.8 upon binding. Benzoate binds to 2-thio-D-amino acid oxidase with a K(,d) of 84 (mu)M. The flavin C(2) = S is not accessible to solvent, reacting with methyl methanethiosulfonate only after dissociation from the protein.Ph.D.BiochemistryUniversity of Michiganhttp://deepblue.lib.umich.edu/bitstream/2027.42/158632/1/8204649.pd