PMID:2567735
| Citation |
Seddon, AP, Zhao, KY and Meister, A (1989) Activation of glutamate by gamma-glutamate kinase: formation of gamma-cis-cycloglutamyl phosphate, an analog of gamma-glutamyl phosphate. J. Biol. Chem. 264:11326-35 |
|---|---|
| Abstract |
gamma-Glutamate kinase, the enzyme that catalyzes the first step in the pathway from glutamate to proline, has been postulated to convert glutamate to a gamma-activated form (possibly gamma-glutamyl phosphate), which is reduced by a NADPH-linked reductase to yield glutamate gamma-semialdehyde (in equilibrium with delta 1-pyrroline-5-carboxylate). In the present work we found that the kinase, in the absence or presence of the reductase (and in the absence of NADPH), catalyzes stoichiometric formation of 5-oxo-L-proline and Pi from L-glutamate and ATP, but catalyzes hydroxamate formation at only about 10% of the rate of ATP-cleavage. A new substrate of the kinase was found; thus, cis-cycloglutamate (cis-1-amino-1,3-dicarboxycyclohexane), a glutamate analog which cannot cyclize to form an analog of 5-oxoproline, interacts effectively with the kinase. The trans form of cycloglutamate does not interact with the kinase; only the cis form can assume a diequatorial conformation equivalent to the extended conformation of glutamate. cis-Cycloglutamyl phosphate formation was shown and evidence was obtained for formation of an enzyme-ADP-cycloglutamyl phosphate complex. Although cis-cycloglutamyl phosphate is not a reducible substrate of the NADPH-dependent reductase, the findings indicate that it interacts with the reductase. These studies, which elucidate several aspects of the mechanism of the utilization of glutamate for formation of delta 1-pyrroline-5-carboxylate, support the hypothesis that the kinase and reductase function as an enzyme complex. A model is suggested in which gamma-glutamyl phosphate formed on the kinase interacts with the reductase to form a gamma-glutamyl-reductase complex, which is reduced by NADPH to yield glutamate gamma-semialdehyde. |
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| Keywords |
Adenosine Diphosphate/metabolism; Adenosine Triphosphate/metabolism; Aldehyde Oxidoreductases/metabolism; Chromatography, High Pressure Liquid; Drug Stability; Escherichia coli/enzymology; Glutamate-5-Semialdehyde Dehydrogenase; Glutamates/metabolism; Glutamic Acid; Hydroxamic Acids/metabolism; Kinetics; Molecular Conformation; NADP/metabolism; Organophosphorus Compounds/metabolism; Phosphates/metabolism; Phosphotransferases/metabolism; Phosphotransferases (Carboxyl Group Acceptor); Pyrroles/metabolism; Pyrrolidonecarboxylic Acid/metabolism |
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