Mechanistic studies on the rat kidney flavoenzyme L-alpha-hydroxy acid oxidase

The falvoenzyme L-alpha-hydroxy acid oxidase from rat kidney [T.H Cromartie and C.T. Walsh (1975), Biochemistry 14, 2588] fails to catalyze the elimination of HCl form D,L-beta-chlorolactate, although this compound is a substrate for oxidation by the enzyme. Deuterium isotope effects demonstrate that proton removal from the alpha carbon of alpha-hydroxy acids is fully rate limiting, a finding in agreement with observations on L-lactate dehydrogenase from yeast [F. Lederer (1974), Eur. J. Biochem. 46, 393] which also does not promote elimination from D,L-beta-chlorolactate. Both D-alpha-hydroxy acid oxidase were found to be rapidly and irreversibly inactivated by the acetylenic substrate 1-hydroxy-3-butynoate. The partially purified dehydrogenase was observed to be inactivated within 10 min by 6.8 times 10(-8) M hydroxybutynoate. For the more extensively studied oxidase, inactivation was found to occur after 25 catalytic events, inactivation occurring by covalent addition of the inactivator to the coenzyme. A stoichimometry of one molecule of hydroxybutynoate per flavine was found, and the time course of inactivation was unaffected by the presence of thiols. The oxidase could also be inactivated by prolonged incubation of the enzyme with 2-hydroxy-3-butenoate, and inactivation which could be completely prevented by the presence of thiolds. Since the inactivation with hydroxybutenoate also left the flavine coenzyme unaltered, the inactivation was attributed to Michael addition of nucleophiles on the enzyme of the ketobutenoate product. Several 4-alkyl-substitued 2-hydroxy-3-butynoates were also observed to inactivate the oxidase by both coenzyme modification and random addition to the apoenzyme. It is proposed that the inactivation may occur by nucleophilic addition of a C4 allenic carbanion to the oxidized flavine coenzyme.