18O studies on the 5-oxoprolinase reaction. Evidence for a phosphorylated tetrahedral intermediate.
5-Oxoprolinase catalyzes a reaction in which the cleavage of ATP to ADP and Pi and the decyclization of 5-oxoproline to form glutamate are coupled. When the reaction catalyzed by 5-oxoprolinase of Pseudomonas putida was carried out to 90% completion in H2(18)O, the residual 5-oxoproline was found to contain 18O in the amide carbonyl oxygen atom. Such isotopic incorporation was not observed in similar studies with a subunit of the enzyme which catalyzes 5-oxoproline-dependent ATPase and formation of a phosphorylated 5-oxoproline intermediate (Seddon, A.P., and Meister, A. (1986) J. Biol. Chem. 261, 11538-11543). When the complete reaction was carried out in H2(18)O, the products glutamate (gamma-carboxyl) and inorganic phosphate were mono- and di-labeled with 18O. Studies with 5-[18O]oxo-L-proline confirmed such replacement of the oxygen atoms of the gamma-carboxyl group of glutamate and the carbonyl oxygen of 5-oxoproline. Oxygen was not transferred from 5-oxoproline to inorganic phosphate. Studies with analogs of 5-oxoproline showed that di-labeling of inorganic phosphate occurred only when ATP hydrolysis was coupled or partially coupled with the decyclization of the substrate. Studies with 5-oxoprolinase from rat kidney gave similar results. These observations are in accord with the view that the reaction involves enzyme-bound phosphorylated intermediates and provide evidence for a phosphorylated tetrahedral intermediate, whose formation is required for coupling.
D015055:1-Carboxyglutamic Acid / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000581:Amidohydrolases / Q000378:metabolism*; D000818:Animals; D007668:Kidney / Q000201:enzymology; D010103:Oxygen Isotopes; D010710:Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011549:Pseudomonas / Q000201:enzymology; D011752:Pyroglutamate Hydrolase / Q000378:metabolism*; D011761:Pyrrolidonecarboxylic Acid / Q000378:metabolism; D051381:Rats
D010103:Oxygen Isotopes; D010710:Phosphates; D010750:Phosphoproteins; D015055:1-Carboxyglutamic Acid; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000581:Amidohydrolases; D011752:Pyroglutamate Hydrolase; D011761:Pyrrolidonecarboxylic Acid