Evidence that a phosphorylated derivative of 5-oxoproline (5-OP) is an intermediate in the reaction catalyzed by 5-oxoprolinase

5-oxo-L-proline + ATP + 2 H2O → L-Glutamate + ADP + P/sub i/ Pseudomonas putida 5-oxoprolinase consists of 2 protein components (A and B). A catalyzes 5-OP-dependent ATPase. B, which does not appear to interact with ATP or 5-OP, is required for catalysis of the complete reaction. Application of the isotope trapping method showed that 5-OP is converted stoichiometrically to an enzyme-bound form in a process requiring ATP and its cleavage. The overall reaction is not inhibited by high concentrations of borohydride. When the reaction catalyzed by A alone is carried out in the presence of 0.1-0.6 M KBH4 there is catalytic formation of proline. Studies with KB3H4 showed incorporation of 3H into proline. Proline formation is prevented by addition of B