Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1981 Dec 1;199(3):603–609. doi: 10.1042/bj1990603

Factors that influence the translocation of the N-carboxybiotin moiety between the two sub-sites of pyruvate carboxylase.

G J Goodall, G S Baldwin, J C Wallace, D B Keech
PMCID: PMC1163416  PMID: 7340821

Abstract

The active site of pyruvate carboxylase, like those of all biotin-dependent carboxylases, is believed to consist of two spatially distinct sub-sites with biotin acting as a mobile carboxy-group carrier oscillating between the two sub-sites. Some of the factors that influence the location and rate of movement of the N-carboxybiotin were studied. The rate of carboxylation of the alternative substrate, 2-oxobutyrate, was measured at 0 degrees C in an assay system where the isolated enzyme--[14C]carboxybiotin was the carboxy-group donor. The results are consistent with the hypothesis that the location of the carboxybiotin in the active site is determined by the presence of Mg2+, acetyl-CoA and the oxo acid substrate. The presence of Mg2+ favours the holding of the complex at the first sub-site, whereas alpha-oxo acids induce the complex to move to the second sub-site. At low concentrations pyruvate induces this movement but does not efficiently act as a carboxy-group acceptor; hydroxypyruvate, glyoxylate and oxamate, though not carboxylated, still induce the movement. The allosteric activator acetyl-CoA exerts only a slight stimulation on the rate of translocation to the second sub-site, and this stimulation arises from an increase in the dissociation constant for Mg2+.

Full text

PDF
608

Selected References

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

  1. Ashman L. K., Keech D. B. Sheep kidney pyruvate carboxylase. Studies on the coupling of adenosine triphosphate hydrolysis and CO2 fixation. J Biol Chem. 1975 Jan 10;250(1):14–21. [PubMed] [Google Scholar]
  2. Bais R., Keech B. The magnesium ion (Mg 2+ ) activation of sheep kidney pyruvate carboxylase. J Biol Chem. 1972 May 25;247(10):3255–3261. [PubMed] [Google Scholar]
  3. Barden R. E., Fung C. H., Utter M. F., Scrutton M. C. Pyruvate carboxylase from chicken liver. Steady state kinetic studies indicate a "two-site" ping-pong mechanism. J Biol Chem. 1972 Feb 25;247(4):1323–1333. [PubMed] [Google Scholar]
  4. Cheung Y. F., Walsh C. Studies on the intramolecular and intermolecular kinetic isotope effects in pyruvate carboxylase catalysis. Biochemistry. 1976 Aug 24;15(17):3749–3754. doi: 10.1021/bi00662a017. [DOI] [PubMed] [Google Scholar]
  5. Easterbrook-Smith S. B., Hudson P. J., Goss N. H., Keech D. B., Wallace J. C. Pyruvate carboxylase: mechanism on the second partial reaction. Arch Biochem Biophys. 1976 Oct;176(2):709–720. doi: 10.1016/0003-9861(76)90215-0. [DOI] [PubMed] [Google Scholar]
  6. Easterbrook-Smith S. B., Wallace J. C., Keech D. B. A reappraisal of the reaction pathway of pyruvate carboxylase. Biochem J. 1978 Jan 1;169(1):225–228. doi: 10.1042/bj1690225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Goss N. H., Dyer P. Y., Keech D. B., Wallace J. C. An electron microscopic study of pyruvate carboxylase. J Biol Chem. 1979 Mar 10;254(5):1734–1739. [PubMed] [Google Scholar]
  8. Helmerhorst E., Stokes G. B. Microcentrifuge desalting: a rapid, quantitative method for desalting small amounts of protein. Anal Biochem. 1980 May 1;104(1):130–135. doi: 10.1016/0003-2697(80)90287-0. [DOI] [PubMed] [Google Scholar]
  9. Mildvan A. S., Scrutton M. C., Utter M. F. Pyruvate carboxylase. VII. A possible role for tightly bound manganese. J Biol Chem. 1966 Aug 10;241(15):3488–3498. [PubMed] [Google Scholar]
  10. Morell A. G., Irvine R. A., Sternlieb I., Scheinberg I. H., Ashwell G. Physical and chemical studies on ceruloplasmin. V. Metabolic studies on sialic acid-free ceruloplasmin in vivo. J Biol Chem. 1968 Jan 10;243(1):155–159. [PubMed] [Google Scholar]
  11. Moss J., Lane M. D. The biotin-dependent enzymes. Adv Enzymol Relat Areas Mol Biol. 1971;35:321–442. doi: 10.1002/9780470122808.ch7. [DOI] [PubMed] [Google Scholar]
  12. Rose I. A., O'Connell E. L., Solomon F. Intermolecular tritium transfer in the transcarboxylase reaction. J Biol Chem. 1976 Feb 10;251(3):902–904. [PubMed] [Google Scholar]
  13. Rétey J., Lynen F. Zur biochemischen Funktion des Biotins. IX. Der sterische Verlauf der Carboxylierung von Propionyl-CoA. Biochem Z. 1965 Aug 6;342(3):256–271. [PubMed] [Google Scholar]
  14. Scrutton M. C., Utter M. F. Pyruvate carboxylase. V. Interaction of the enzyme with adenosine triphosphate. J Biol Chem. 1965 Oct;240(10):3714–3723. [PubMed] [Google Scholar]
  15. Stubbe J., Fish S., Abeles R. H. Are carboxylations involving biotin concerted or nonconcerted? J Biol Chem. 1980 Jan 10;255(1):236–242. [PubMed] [Google Scholar]
  16. Warren G. B., Tipton K. F. Pig liver pyruvate carboxylase. The reaction pathway for the carboxylation of pyruvate. Biochem J. 1974 May;139(2):311–320. doi: 10.1042/bj1390311. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES