5-Oxo-L-prolinase (L-pyroglutamate hydrolase). Studies of the chemical mechanism.
Rat kidney 5-oxo-L-prolinase catalyzes the endergonic hydrolysis of 5-oxo-L-proline (L-pyroglutamate, L-2-pyrrolidone-5-carboxylate) to form L-glutamate; the reaction is driven by and dependent on the stoichiometric concomitant hydrolysis of ATP to ADP and inorganic phosphate. The present studies are concerned with the mechanism by which the free energy of ATP hydrolysis is conserved and made available for 5-oxoproline hydrolysis. Studies with 18O-labeled substrates showed that (a) all three oxygen atoms of 5-oxoproline are recovered in the product glutamate, and (b) the two water molecules consumed in the reaction contribute one oxygen atom to inorganic phosphate and one oxygen atom to the gamma-carboxyl group of glutamate. It was shown that the enzyme also catalyzes the intrinsically exergonic hydrolysis of alpha-hydroxyglutarate lactone, a reaction that is ATP-dependent. Intermediates in the 5-oxoprolinase reaction were not detected by exchange experiments with radioactive ADP and phosphate, nor were they trapped by adding hydroxylamine. In the presence of very high glutamate concentrations, a slow reversal of the 5-oxoprolinase reaction was demonstrated by measuring ATP formation. The findings are consistent with a mechanism in which 5-oxo-L-proline is phosphorylated by ATP on the amide carbonyl oxygen and the resulting intermediate is subsequently hydrolyzed to yield gamma-glutamyl phosphate; the latter is hydrolyzed to glutamate and inorganic phosphate.
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000581:Amidohydrolases / Q000378:metabolism*; D000818:Animals; D005971:Glutamates; D018698:Glutamic Acid; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D010103:Oxygen Isotopes; D011752:Pyroglutamate Hydrolase / Q000378:metabolism*; D051381:Rats
D005971:Glutamates; D010103:Oxygen Isotopes; D018698:Glutamic Acid; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000581:Amidohydrolases; D011752:Pyroglutamate Hydrolase