Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:6.3.5.5 (CPS)
 1,262 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the course of studies on glutamine-dependent carbamyl phosphate synthetase from Aerobacter aerogenes, we purified another protein which was found to be glutamate synthase (EC 2.6.1.53). The enzyme, obtained in apparently homogeneous form (monomer molecular weight about 227,000; s(20,omega) = 17.6 S), was found to be a typical glutamine amidotransferase in that it exhibits glutaminase activity and can utilize ammonia in place of glutamine as a nitrogen donor. The enzyme also catalyzes at low rates the oxidative deamination of glutamate in the presence of TPN, and it exhibits TPNH oxidase activity. The enzyme is similar to the glutamate synthase found in Escherichia coli in that it is an iron-sulfide flavoprotein. Treatment of the enzyme with sodium dodecyl sulfate or potassium thiocyanate dissociates it into nonidentical subunits exhibiting molecular weights of about 175,000 and 51,500. The glutamine-dependent activity of the enzyme is inhibited by L-2-amino-4-oxo-5-chloropentanoic acid, but this chloroketone analog of glutamine does not affect the ammonia-dependent glutamate synthase activity. Studies with [(14)C]chloroketone show that the reagent binds to the heavy subunit only. Inhibition by the chloroketone and its binding to the heavy subunit are markedly reduced in the presence of L-glutamine. Sedimentation velocity studies carried out in potassium thiocyanate indicate that iron-sulfide and flavin sites are also located on the heavy subunit. While these studies show that glutamate synthase resembles other glutamine amidotransferases in certain of its catalytic properties, the findings indicate that the light subunit of this enzyme, in contrast to that of several other glutamine amidotransferases, does not function to bind glutamine. It is of interest that the enzyme exhibits an unusually high affinity for ammonia as compared to a number of other glutamine amidotransferases. Glutamate synthase is inhibited (competitively with respect to glutamine) by low concentrations of methionine sulfone, methionine sulfoximine, and methionine sulfoxide.
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PMID:Glutamine-binding subunit of glutamate synthase and partial reactions catalyzed by this glutamine amidotransferase. 453 Oct 4

As a result of recent advances in molecular cloning, protein expression, and X-ray crystallography, it has now become feasible to examine complicated protein structures at high resolution. For those enzymes with multiple catalytic sites, a common theme is beginning to emerge; the existence of molecular tunnels that connect one active site with another. The apparent mechanistic advantages rendered by these molecular conduits include the protection of unstable intermediates and an improvement in catalytic efficiency by blocking the diffusion of intermediates into the bulk solvent. Since the first molecular tunnel within tryptophan synthase was discovered in 1988, tunnels within carbamoyl phosphate synthetase, glutamine phosphoribosylpyrophosphate amidotransferase, asparagine synthetase, glutamate synthase, imidazole glycerol phosphate synthase, glucosamine 6-phosphate synthase, and carbon monoxide dehydrogenase/acetyl-CoA synthase have been identified. The translocation of ammonia, derived from the hydrolysis of glutamine, is the most abundant functional requirement for a protein tunnel identified thus far. Here we describe and summarize our current understanding of molecular tunnels observed in various enzyme systems.
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PMID:Enzymes with molecular tunnels. 1285 15

The primary nitrogen metabolism of the N2-fixing root nodule symbiosis Alnus incana (L.)- Frankia was investigated by 31P and 15N nuclear magnetic resonance (NMR) spectroscopy. Perfusion of root nodules in a pulse-chase approach with 15N- or 14N-labeled NH4+ revealed the presence of the amino acids alanine (Ala), gamma-amino butyric acid, glutamine (Gln), glutamic acid (Glu), citrulline (Cit) and arginine (Arg). Labeling kinetics of the Gln amide-N and alpha-amino acids suggested that the glutamine synthetase (GS; EC 6.3.1.2)-glutamate synthase (GOGAT; EC 1.4.1.13) pathway was active. Inhibition of the GS-catalyzed reaction by methionine sulphoximine abolished incorporation of 15N. Cit was labeled in all three N positions but most rapidly in the omega position, consistent with carbamoyl phosphate as the precursor to which Gln could be the amino donor catalyzed by carbamoyl phosphate synthase (CPS; EC 6.3.5.5). Ala biosynthesis occurred consistent with a flux of N in the sequence Gln-Glu-Ala. 31P NMR spectroscopy in vivo and of extracts revealed several metabolites and was used in connection with the 15N pulse-chase experiment to assess general metabolic status. Stable concentrations of ATP and UDP-glucose during extended perfusions showed that the overall root nodule metabolism appeared undisturbed throughout the experiments. The metabolic pathways suggested by the NMR results were confirmed by high activities of the enzymes GS, NADH-GOGAT and ornithine carbamoyltransferase (OCT; EC 2.1.3.3). We conclude that the primary pathway of NH4+ assimilation in A. incana root nodules occurs through the GS-GOGAT pathway. Biosynthesis of Cit through GS-CPS-OCT is important and is a link between the first amino acid Gln and this final transport and storage form of nitrogen.
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PMID:Primary metabolism in N2-fixing Alnus incana-Frankia symbiotic root nodules studied with 15N and 31P nuclear magnetic resonance spectroscopy. 1517 12

This study was aimed at investigating the physiological role of ferredoxin-glutamate synthases (EC 1.4.1.7), NADH-glutamate synthase (EC 1.4.1.14) and carbamoylphosphate synthetase (EC 6.3.5.5) in Arabidopsis. Phenotypic analysis revealed a high level of photorespiratory ammonium, glutamine/glutamate and asparagine/aspartate in the GLU1 mutant lacking the major ferredoxin-glutamate synthase, indicating that excess photorespiratory ammonium was detoxified into amino acids for transport out of the veins. Consistent with these results, promoter analysis and in situ hybridization demonstrated that GLU1 and GLU2 were expressed in the mesophyll and phloem companion cell-sieve element complex. However, these phenotypic changes were not detected in the GLU2 mutant defective in the second ferredoxin-glutamate synthase gene. The impairment in primary ammonium assimilation in the GLT mutant under nonphotorespiratory high-CO(2) conditions underlined the importance of NADH-glutamate synthase for amino acid trafficking, given that this gene only accounted for 3% of total glutamate synthase activity. The excess ammonium from either endogenous photorespiration or the exogenous medium was shifted to arginine. The promoter analysis and slight effects on overall arginine synthesis in the T-DNA insertion mutant in the single carbamoylphosphate synthetase large subunit gene indicated that carbamoylphosphate synthetase located in the chloroplasts was not limiting for ammonium assimilation into arginine. The data provided evidence that ferredoxin-glutamate synthases, NADH-glutamate synthase and carbamoylphosphate synthetase play specific physiological roles in ammonium assimilation in the mesophyll and phloem for the synthesis and transport of glutamine, glutamate, arginine, and derived amino acids.
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PMID:Assimilation of excess ammonium into amino acids and nitrogen translocation in Arabidopsis thaliana--roles of glutamate synthases and carbamoylphosphate synthetase in leaves. 1955 10