5-Oxyoprolinase: Structure and mechanism of action

5-Oxoprolinase catalyzes the endergonic cleavage of 5-oxo-L-proline to L-glutamate, coupled to the exergonic hydrolysis of ATP to ADP and Pi. In the present studies, the enzyme present in a strain of Pseudomonas putida was found to be composed of two protein components. Component A catalyzes 5-oxo-L-proline-dependent cleavage of ATP without 5-oxyoproline decyclization. Component B is required to couple ATP hydrolysis to the ring-opening of 5-oxoproline. The two components have been purified to apparent homogeneity. When the 5-oxoprolinase reactions were carried out to 90% completion in H218O, the residual 5-oxoproline contained 18O in the amide carbonyl group. Glutamate and Pi isolated from the complete reactions conducted in H218O were mono- and di-labeled with the isotope. No isotope was incorporated into 5-oxoproline if Component A was used alone. Studies with 5-oxoproline analogs showed that dilabeling of Pi occurred only in coupled or partially coupled reactions. Using 5-[18O]oxo-L-[5-13C]proline, the reactions were further analyzed by 13C-NMR. 5-Oxoprolinase from rat kidney was used as a comparison in all mechanistic studies, and similar results were obtained. These findings are consistent with the view that the reaction involves enzyme-bound, phosphorylated intermediates, and provided strong evidence for a phosphorylated tetrahedral intermediate, the formation of which is required for the coupling