Activation of glutamate by gamma-glutamate kinase: formation of gamma-cis-cycloglutamyl phosphate, an analog of gamma-glutamyl phosphate.
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.
Seddon
A P
AP
Department of Biochemistry, Cornell University Medical College, New York, New York 10021.
Zhao
K Y
KY
Meister
A
A
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000445:Aldehyde Oxidoreductases / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004355:Drug Stability; D004926:Escherichia coli / Q000201:enzymology*; D050698:Glutamate-5-Semialdehyde Dehydrogenase; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D006877:Hydroxamic Acids / Q000378:metabolism; D007700:Kinetics; D008968:Molecular Conformation; D009249:NADP / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D010770:Phosphotransferases / Q000378:metabolism*; D017851:Phosphotransferases (Carboxyl Group Acceptor); D011758:Pyrroles / Q000378:metabolism; D011761:Pyrrolidonecarboxylic Acid / Q000378:metabolism
D005971:Glutamates; D006877:Hydroxamic Acids; D009943:Organophosphorus Compounds; D010710:Phosphates; D011758:Pyrroles; C060122:gamma-cycloglutamyl phosphate; C015485:delta-1-pyrroline-5-carboxylate; D018698:Glutamic Acid; D009249:NADP; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000445:Aldehyde Oxidoreductases; D050698:Glutamate-5-Semialdehyde Dehydrogenase; D010770:Phosphotransferases; D017851:Phosphotransferases (Carboxyl Group Acceptor); C020650:glutamate 5-kinase; D011761:Pyrrolidonecarboxylic Acid