STUDIES OF MECHANISM OF ACTION OF D-PROLINE REDUCTASE - PRESENCE ON COVALENTLY BOUND PYRUVATE AND ITS ROLE IN CATALYTIC PROCESS

When a partially purified preparation of d-proline reductase reacts with hydroxylamine, phenylhydrazine, and NaBH4, the enzymes becomes inactivated, suggesting that a carbonyl group is required for catalytic activity. In the presence of mercaptans the enzyme is inactivated by alkylating agents. When the enzyme reacts with NaBH4(3H), nonexchangeable tritium is introduced into the protein. Acid hydrolysis of the tritiated protein releases labeled lactate and enzymic hydrolysis produces fragments electrophoretically distinguishable from reduced enzyme, which contain covalently bound labeled lactate. Alkaline hydrolysis of the enzyme releases a fragment which is not lactate, but from which lactate can be released by acid hydrolysis. These results establish the presence of pyruvate covalently bound to a protein. We consider it highly probable that the carbonyl group of pyruvate is involved in the catalytic activity. It is tentatively proposed that an adduct is found between the substrate nitrogen and the carbonyl group of the enzyme. Formation of this adduct enhances the leaving ability of the nitrogen and facilitates the reductive cleavage of the carbon-nitrogen bond. © 1969.