Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1974 Nov;71(11):4607–4611. doi: 10.1073/pnas.71.11.4607

Glutamine-Binding Subunit of Glutamate Synthase and Partial Reactions Catalyzed by This Glutamine Amidotransferase

Paul P Trotta 1, Karen E B Platzer 1, Rudy H Haschemeyer 1, Alton Meister 1
PMCID: PMC433937  PMID: 4531004

Abstract

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; s20,ω = 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 [14C]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.

Keywords: enzyme dissociation, flavin, iron-sulfide enzyme, L-2-amino-4-oxo-5-chloropentanoate

Full text

PDF
4607

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Brown C. M., Burn V. J., Johnson B. Presence of glutamate synthase in fission yeasts and its possible role in ammonia assimilation. Nat New Biol. 1973 Nov 28;246(152):115–116. doi: 10.1038/newbio246115a0. [DOI] [PubMed] [Google Scholar]
  2. DAVIS B. J. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. doi: 10.1111/j.1749-6632.1964.tb14213.x. [DOI] [PubMed] [Google Scholar]
  3. Elmerich C., Aubert J. P. Synthesis of glutamate by a glutamine: 2-oxo-glutarate amidotransferase (NADP oxidoreductase) in Bacillus megaterium. Biochem Biophys Res Commun. 1971 Feb 5;42(3):371–376. doi: 10.1016/0006-291x(71)90380-9. [DOI] [PubMed] [Google Scholar]
  4. Horowitz B., Meister A. Glutamine-dependent asparagine synthetase from leukemia cells. Chloride dependence, mechanism of action, and inhibition. J Biol Chem. 1972 Oct 25;247(20):6708–6719. [PubMed] [Google Scholar]
  5. Ito J., Yanofsky C. The nature of the anthranilic acid synthetase complex of Escherichia coli. J Biol Chem. 1966 Sep 10;241(17):4112–4114. [PubMed] [Google Scholar]
  6. Kane J. F., Holmes W. M., Jensen R. A. Metabolic interlock. The dual function of a folate pathway gene as an extra-operonic gene of tryptophan biosynthesis. J Biol Chem. 1972 Mar 10;247(5):1587–1596. [PubMed] [Google Scholar]
  7. Khedouri E., Anderson P. M., Meister A. Selective inactivation of the glutamine binding site of Escherichia coli carbamyl phosphate synthetase by 2-amino-4-oxo-5-chloropentanoic acid. Biochemistry. 1966 Nov;5(11):3552–3557. doi: 10.1021/bi00875a024. [DOI] [PubMed] [Google Scholar]
  8. LOVENBERG W., BUCHANAN B. B., RABINOWITZ J. C. STUDIES ON THE CHEMICAL NATURE OF CLOSTRIDIAL FERREDOXIN. J Biol Chem. 1963 Dec;238:3899–3913. [PubMed] [Google Scholar]
  9. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  10. Meers J. L., Tempest D. W., Brown C. M. 'Glutamine(amide):2-oxoglutarate amino transferase oxido-reductase (NADP); an enzyme involved in the synthesis of glutamate by some bacteria. J Gen Microbiol. 1970 Dec;64(2):187–194. doi: 10.1099/00221287-64-2-187. [DOI] [PubMed] [Google Scholar]
  11. Miller R. E., Stadtman E. R. Glutamate synthase from Escherichia coli. An iron-sulfide flavoprotein. J Biol Chem. 1972 Nov 25;247(22):7407–7419. [PubMed] [Google Scholar]
  12. Nagatani H., Shimizu M., Valentine R. C. The mechanism of ammonia assimilation in nitrogen fixing Bacteria. Arch Mikrobiol. 1971;79(2):164–175. doi: 10.1007/BF00424923. [DOI] [PubMed] [Google Scholar]
  13. Pinkus L. M., Meister A. Identification of a reactive cysteine residue at the glutamine binding site of carbamyl phosphate synthetase. J Biol Chem. 1972 Oct 10;247(19):6119–6127. [PubMed] [Google Scholar]
  14. Roon R. J., Even H. L., Larimore F. Glutamate synthase: properties of the reduced nicotinamide adenine dinucleotide-dependent enzyme from Saccharomyces cerevisiae. J Bacteriol. 1974 Apr;118(1):89–95. doi: 10.1128/jb.118.1.89-95.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Tempest D. W., Meers J. L., Brown C. M. Synthesis of glutamate in Aerobacter aerogenes by a hitherto unknown route. Biochem J. 1970 Apr;117(2):405–407. doi: 10.1042/bj1170405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Trotta P. P., Estis L. F., Meister A., Haschemeyer R. H. Self-association and allosteric properties of glutamine-dependent carbamyl phosphate synthetase. Reversible dissociation to monomeric species. J Biol Chem. 1974 Jan 25;249(2):482–489. [PubMed] [Google Scholar]
  17. Trotta P. P., Pinkus L. M., Haschemeyer R. H., Meister A. Reversible dissociation of the monomer of glutamine-dependent carbamyl phosphate synthetase into catalytically active heavy and light subunits. J Biol Chem. 1974 Jan 25;249(2):492–499. [PubMed] [Google Scholar]
  18. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES