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. 1985 Jan 1;225(1):209–217. doi: 10.1042/bj2250209

Negative co-operativity in glutamate dehydrogenase. Involvement of the 2-position in glutamate in the induction of conformational changes.

E T Bell, C LiMuti, C L Renz, J E Bell
PMCID: PMC1144571  PMID: 2858197

Abstract

The 2-position substituent on substrates or substrate analogues for glutamate dehydrogenase is shown to be intimately involved in the induction of conformational changes between subunits in the hexamer by coenzyme. These conformational changes are associated with the negative co-operativity exhibited by this enzyme. 2-Oxoglutarate and L-2-hydroxyglutarate induce indications of co-operativity similar to those induced by the substrate of oxidative deamination, glutamate, in kinetic studies. Glutarate (2-position CH2) does not. A comparison of the effects of L-2-hydroxyglutarate and D-2-hydroxyglutarate or D-glutamate indicates that the 2-position substituent must be in the L-configuration for these conformational changes to be triggered. In addition, glutarate and L-glutamate in ternary enzyme-NAD(P)H-substrate complexes induce very different coenzyme fluorescence properties, showing that glutamate induces a different conformation of the enzyme-coenzyme complex from that induced by glutarate. Although glutamate and glutarate both tighten the binding of reduced coenzyme to the active site, the effect is much greater with glutamate, and the binding is described by two dissociation constants when glutamate is present. The data suggest that the two carboxy groups on the substrate are required to allow synergistic binding of coenzyme and substrate to the active site, but that interactions between the 2-position on the substrate and the enzyme trigger the conformational changes that result in subunit-subunit interactions and in the catalytic co-operativity exhibited by this enzyme.

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Selected References

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

  1. Alex S., Bell J. E. Dual nucleotide specificity of bovine glutamate dehydrogenase. The role of negative co-operativity. Biochem J. 1980 Nov 1;191(2):299–304. doi: 10.1042/bj1910299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bell J. E., Dalziel K. A conformational transition of the oligomer of glutamate dehydrogenase induced by half-saturation with NAD + or NADP + . Biochim Biophys Acta. 1973 May 5;309(1):237–242. doi: 10.1016/0005-2744(73)90336-7. [DOI] [PubMed] [Google Scholar]
  3. CAUGHEY W. S., HELLERMAN L., SMILEY J. D. L-glutamic acid dehydrogenase; structural requirements for substrate competition; effect of thyroxine. J Biol Chem. 1957 Jan;224(1):591–607. [PubMed] [Google Scholar]
  4. Chen S. S., Engel P. C. The allosteric mechanism of bovine liver glutamate dehydrogenase. Evidence from circular-dichroism studies for a conformational change in the ternary complex enzyme-(oxidized nicotinamide-adenine dinucleotide)-glutarate. Biochem J. 1977 May 1;163(2):297–302. doi: 10.1042/bj1630297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen S., Engel P. C. Protection of glutamate dehydrogenase by nicotinamide-adenine dinucleotide against reversible inactivation by pyridoxal 5'-phosphate as a sensitive indicator of conformational change induced by substrates and substrate analogues. Biochem J. 1974 Dec;143(3):569–574. doi: 10.1042/bj1430569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dalziel K., Egan R. R. The binding of oxidized coenzymes by glutamate dehydrogenase and the effects of glutarate and purine nucleotides. Biochem J. 1972 Feb;126(4):975–984. doi: 10.1042/bj1260975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dalziel Keith, Engel Paul C. Antagonistic homotropic interactions as a possible explanation of coenzyme activation of glutamate dehydrogenase. FEBS Lett. 1968 Oct;1(5):349–352. doi: 10.1016/0014-5793(68)80153-x. [DOI] [PubMed] [Google Scholar]
  8. Egan R. R., Dalziel K. Active centre equivalent weight of glutamate dehydrogenase from dry weight determinations and spectrophotometric titrations of abortive complexes. Biochim Biophys Acta. 1971 Oct;250(1):47–50. doi: 10.1016/0005-2744(71)90118-5. [DOI] [PubMed] [Google Scholar]
  9. Engel P. C., Chen S. S. A product-inhibition study of bovine liver glutamate dehydrogenase. Biochem J. 1975 Nov;151(2):305–318. doi: 10.1042/bj1510305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Engel P. C., Dalziel K. Kinetic studies of glutamate dehydrogenase with glutamate and norvaline as substrates. Coenzyme activation and negative homotropic interactions in allosteric enzymes. Biochem J. 1969 Dec;115(4):621–631. doi: 10.1042/bj1150621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Engel P. C., Ferdinand W. The significance of abrupt transitions in Lineweaver-Burk plots with particular reference to glutamate dehydrogenase. Negative and positive co-operativity in catalytic rate constants. Biochem J. 1973 Jan;131(1):97–105. doi: 10.1042/bj1310097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. FRIEDEN C. Glutamic dehydrogenase. III. The order of substrate addition in the enzymatic reaction. J Biol Chem. 1959 Nov;234:2891–2896. [PubMed] [Google Scholar]
  13. George A., Bell J. E. Effects of adenosine 5'-diphosphate on bovine glutamate dehydrogenase: diethyl pyrocarbonate modification. Biochemistry. 1980 Dec 23;19(26):6057–6061. doi: 10.1021/bi00567a017. [DOI] [PubMed] [Google Scholar]
  14. Hornby D. P., Engel P. C., Hatanaka S. Beef liver glutamate dehydrogenase: a study of the oxidation of various alternative amino acid substrates retaining the correct spacing of the two carboxylate groups. Int J Biochem. 1983;15(4):495–500. doi: 10.1016/0020-711x(83)90122-2. [DOI] [PubMed] [Google Scholar]
  15. LiMuti C., Bell J. E. A steady-state random-order mechanism for the oxidative deamination of norvaline by glutamate dehydrogenase. Biochem J. 1983 Apr 1;211(1):99–107. doi: 10.1042/bj2110099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Rife J. E., Cleland W. W. Kinetic mechanism of glutamate dehydrogenase. Biochemistry. 1980 May 27;19(11):2321–2328. doi: 10.1021/bi00552a007. [DOI] [PubMed] [Google Scholar]
  17. Rogers K. S., Boots M. R., Boots S. G. Molecular interactions of six aromatic competitive inhibitors with bovine liver glutamate dehydrogenase. Biochim Biophys Acta. 1972 Feb 28;258(2):343–350. doi: 10.1016/0005-2744(72)90225-2. [DOI] [PubMed] [Google Scholar]
  18. Rogers K. S. Molecular interactions of competitive inhibitors with bovine liver glutamate dehydrogenase. J Biol Chem. 1971 Apr 10;246(7):2004–2009. [PubMed] [Google Scholar]
  19. STEIN A. M., LEE J. K., ANDERSON C. D., ANDERSON B. M. THE THIONICOTINAMIDE ANALOGS OF DPN AND TPN. I. PREPARATION AND ANALYSIS. Biochemistry. 1963 Sep-Oct;2:1015–1017. doi: 10.1021/bi00905a018. [DOI] [PubMed] [Google Scholar]
  20. Smith T., Bell J. E. Mechanism of hysteresis in bovine glutamate dehydrogenase: role of subunit interactions. Biochemistry. 1982 Feb 16;21(4):733–737. doi: 10.1021/bi00533a023. [DOI] [PubMed] [Google Scholar]

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