Trapping of an intermediate in the reaction catalyzed by 5-oxoprolinase.
Bacterial 5-oxoprolinase is composed of two protein components: Component A, which catalyzes 5-oxoproline-dependent ATP-hydrolysis and Component B, which couples the hydrolysis of ATP with the decyclization of 5-oxoproline to form glutamate (Seddon, A. P., Li, L., and Meister, A. (1984) J. Biol. Chem. 259, 8091-8094). Studies on this unusual enzyme system have led to evidence that an intermediate is formed by Component A. Application of the isotope-trapping method demonstrated an activated 5-oxoproline intermediate, whose formation requires ATP, Mg2+, and Component A. The amount of ATP-dependent trapping was close to the number of enzyme active sites. The intermediate formed by Component A was shown to be reducible by potassium borohydride to proline in low yield; when Component B was added, the formation of proline was abolished. Treatment of reaction mixtures containing Component A, 5-oxoproline, and [gamma-32P] ATP with diazomethane led to appearance of a 32P-labeled compound (found on thin layer chromatography), whose formation was significantly reduced when Component B was present. The new compound, which is labile, breaks down to form dimethyl[32P]phosphate. The total amount of dimethyl[32P]phosphate formed after breakdown is close to the number of active sites of Component A. The data are consistent with the conclusion that a phosphorylated form of 5-oxoproline is formed by Component A and suggest that Component B is required for conversion of this intermediate to glutamate.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000581:Amidohydrolases / Q000378:metabolism*; D001894:Borohydrides / Q000378:metabolism; D002250:Carbon Radioisotopes; D007700:Kinetics; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011392:Proline / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology; D011752:Pyroglutamate Hydrolase / Q000378:metabolism*; D014316:Tritium
D001894:Borohydrides; D002250:Carbon Radioisotopes; D010761:Phosphorus Radioisotopes; C028371:potassium borohydride; D014316:Tritium; D000255:Adenosine Triphosphate; D011392:Proline; D000581:Amidohydrolases; D011752:Pyroglutamate Hydrolase