5-Oxo-L-prolinase (L-pyroglutamate hydrolase). Purification and catalytic properties.
5-Oxo-L-prolinase, an enzyme that catalyzes the conversion of 5-oxo-L-proline (L-pyroglutamate; L-2-pyrrolidone-5-carboxylate) to L-glutamate coupled with the cleavage of ATP to ADP and Pi, has been purified about 1600-fold from rat kidney. Purification was carried out in the presence of 5-oxo-L-proline which protects the enzyme under a variety of conditions. An estimate of the molecular weight (about 325,000) was made by gel filtration on Sephadex G-200. K+ (or NH4+) and Mg2+ were required for activity. GTP, ITP, CTP, and UTP were much less active than ATP; dATP was 43% as active as ATP. ADP inhibited and addition of pyruvate kinase and phosphoenolpyruvate activated the reaction. The enzyme, which is protected during storage by dithiothreitol, is inhibited by p-hydroxymercuribenzoate, N-ethylmaleimide, and iodoacetamide. The apparent Km values for 5-oxo-L-proline and ATP are, respectively, 0.05 and 0.17 mM. The pH profile indicates a broad range of activity from about pH 5.5 to pH 11.2 with apparent maxima at about pH 7 and pH 9.7. The formation of Pi and glutamate was equimolar over a wide pH range. When the enzyme was incubated with ATP, Mg2+, K+, and L-2-imidazolidone-4-carboxylate or L-dihydroorotate, cleavage of ATP to ADP and Pi occurred, but no cleavage of the imino acid substrates was observed; when the enzyme was incubated under these conditions with 2-piperidone-6-carboxylate, 4-oxy-5-oxoproline, and 3-oxy-5-oxoproline, the corresponding dicarboxylic amino acids were formed, but the molar ratio of Pi to amino acid formation was significantly greater than unity.
D000581:Amidohydrolases / Q000302:isolation & purification*; D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008297:Male; D008928:Mitochondria / Q000201:enzymology; D008970:Molecular Weight; D009928:Organ Specificity; D011392:Proline / Q000031:analogs & derivatives; D011489:Protein Denaturation; D011752:Pyroglutamate Hydrolase / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions / Q000201:enzymology
D011392:Proline; D000581:Amidohydrolases; D011752:Pyroglutamate Hydrolase