18O-studies on the mechanism of action of 5-oxoprolinase

When the reaction catalyzed by bacterial 5-oxoprolinase was carried out to 80% of completion in 18O-enriched H2O, 18O was found in the P/sub i/ and glutamate (γ-COOH) products and also in amide carbonyl oxygen of the residual 5-oxoproline. Some glutamate molecules contained 2 atoms of 18O (γ-COOH). In similar studies with component A alone, 18O was incorporated into P/sub i/ but not into 5-oxoproline. When enzyme isolated from rat kidney was run in 18O-enriched H2O, isotope was found in the residual 5-oxoproline, glutamate and P/sub i/. Some glutamate contained 2 γ-carboxyl 18O atoms and di-labeling of P/sub i/ was also observed. The results obtained with 5-oxoprolinase isolated from kidney and bacteria are thus comparable. The reversibility of the reaction catalyzed by the bacterial enzyme (A + B) was carefully examined and no reversibility was observed. The labeling patterns observed are consistent with a mechanism in which the phosphorylated enol form of 5-oxoproline is hydrated and converted to γ-glutamyl phosphate which is hydrolyzed at phosphate to give glutamate. A detailed mechanism consistent with these data will be presented