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. 1975 May;147(2):351–358. doi: 10.1042/bj1470351

The equilibrium position of the reaction of bovine liver glutamate dehydrogenase with pyridoxal5'-phosphate. A demonstration that covalent modification with this reagent completely abolishes catalytic activity.

S S Chen, P C Engel
PMCID: PMC1165449  PMID: 1237292

Abstract

1. The activity of bovine liver glutamate dehydrogenase incubated with pyridoxal 5'-phosphate declined to a steady value reached within 30--60 min. The residual activity depended on the concentration of modifier up to about 5 mM. Above this concentration, however, no further inactivation was produced. The minimum activity obtainable in such incubations was 6--7% of the initial value. 2. Km values of the modified enzyme were unaltered, whereas Vmax. was decreased. 3. Activity was fully regained on dialysis against 0.1 M-potassium phosphate buffer. 4. Reduction with borohydride rendered the inactivation permanent but did not alter its extent. 5. Enzyme permanently inactivated in this way to the extent of 90% and dialysed was re-treated with pyridoxal 5'-phosphate. In this second cycle activity declined from 10 to 1% of the original activity. 6. This strongly suggests that the failure to achieve complete inactivation in a single cycle reflects a reversible equilibrium between inactive Schiff base, i.e. covalently modified enzyme, and a non-covalent complex. 7. The re-inactivation reaction occurring on dilution was demonstrated directly and a first-order rate constant obtained (0.048 min-1). This, in conjunction with an estimate of the forward rate constant for Schiff-base formation, obtained by approximate pseudo-first-order analysis of inactivation at varied modifier concentrations, gives a predicted minimum activity very close to that actually obtained in a single cycle of treatment. 8. The dissociation constant of the non-covalent complex is given by two methods as 0.90 and 1.59mM. 9. The results indicate that covalent modification with pyridoxal 5'-phosphate completely abolishes the activity of glutamate dehydrogenase.

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Selected References

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

  1. Anderson B. M., Anderson C. D., Churchich J. E. Inhibition of glutamic dehydrogenase by pyridoxal 5'-phosphate. Biochemistry. 1966 Sep;5(9):2893–2900. doi: 10.1021/bi00873a017. [DOI] [PubMed] [Google Scholar]
  2. Brown A., Culver J. M., Fisher H. F. Mechanism of inactivation of L-glutamate dehydrogenase by pyridoxal and pyridoxal phosphate. Biochemistry. 1973 Oct 23;12(22):4367–4373. doi: 10.1021/bi00746a011. [DOI] [PubMed] [Google Scholar]
  3. Coffee C. J., Bradshaw R. A., Goldin B. R., Frieden C. Identification of the sites of modification of bovine liver glutamate dehydrogenase reacted with trinitrobenzenesulfonate. Biochemistry. 1971 Sep 14;10(19):3516–3526. doi: 10.1021/bi00795a005. [DOI] [PubMed] [Google Scholar]
  4. DALZIEL K. Kinetic studies of liver alcohol dehydrogenase. Biochem J. 1962 Aug;84:244–254. doi: 10.1042/bj0840244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Di Prisco G. Tyrosyl and lysyl residues involved in the reactivity of catalytic and regulatory sites of crystalline beef liver glutamate dehydrogenase. Biochemistry. 1971 Feb 16;10(4):585–589. doi: 10.1021/bi00780a007. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Goldin B. R., Frieden C. The effect of pyridoxal phosphate modification on the catalytic and regulatory properties of bovine liver glutamate dehydrogenase. J Biol Chem. 1972 Apr 10;247(7):2139–2144. [PubMed] [Google Scholar]
  9. Holbrook J. J., Jeckel R. A peptide containing a reactive lysyl group from ox liver glutamate dehydrogenase. Biochem J. 1969 Mar;111(5):689–694. doi: 10.1042/bj1110689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Holbrook J. J., Roberts P. A., Wallis R. B. The site at which 4-iodoacetamidosalicylate reacts with glutamate dehydrogenases. Biochem J. 1973 May;133(1):165–171. doi: 10.1042/bj1330165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Malcolm A. D., Radda G. K. Allosteric transitions of glutamate dehydrogenase. Nature. 1968 Aug 31;219(5157):947–949. doi: 10.1038/219947a0. [DOI] [PubMed] [Google Scholar]
  12. Malcolm A. D., Radda G. K. The reaction of glutamate dehydrogenase with 4-iodoacetamido salicylic acid. Eur J Biochem. 1970 Sep;15(3):555–561. doi: 10.1111/j.1432-1033.1970.tb01040.x. [DOI] [PubMed] [Google Scholar]
  13. McKinley-McKee J. S., Morris D. L. The lysines in liver alcohol dehydrogenase. Chemical modification with pyridoxal 5'-phosphate and methyl picolinimidate. Eur J Biochem. 1972 Jun 23;28(1):1–11. doi: 10.1111/j.1432-1033.1972.tb01877.x. [DOI] [PubMed] [Google Scholar]
  14. Piszkiewicz D., Landon M., Smith E. L. Bovine liver flutamate dehydrogenase. Sequence of a hexadecapeptide containing a lysyl residue reactive with pyridoxal 5'-phosphate. J Biol Chem. 1970 May 25;245(10):2622–2626. [PubMed] [Google Scholar]
  15. Piszkiewicz D., Smith E. L. Bovine liver glutamate dehydrogenase. Equilibria and kinetics of inactivation by pyridoxal. Biochemistry. 1971 Nov 23;10(24):4538–4544. doi: 10.1021/bi00800a030. [DOI] [PubMed] [Google Scholar]
  16. Veronese F. M., Piszkiewicz D., Smith E. L. Inactivation of bovine glutamate dehydrogenase by carbamyl phosphate and cyanate. J Biol Chem. 1972 Feb 10;247(3):754–759. [PubMed] [Google Scholar]
  17. Wallis R. B., Holbrook J. J. The effect of modifying lysine-126 on the physical, catalytic and regulatory properties of bovine liver glutamate dehydrogenase. Biochem J. 1973 May;133(1):173–182. doi: 10.1042/bj1330173. [DOI] [PMC free article] [PubMed] [Google Scholar]

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