Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1979 Nov 1;183(2):247–254. doi: 10.1042/bj1830247

Wheat-germ aspartate transcarbamoylase. Steady-state kinetics and stereochemistry of the binding site for L-aspartate.

J E Grayson, R J Yon, P J Butterworth
PMCID: PMC1161553  PMID: 534495

Abstract

1. The steady-state kinetics of the bisubstrate reaction catalysed by aspartate transcarbamoylase purified from wheat (Triticum vulgare)-germ have been studied at 25 degrees C, pH 8.5 AND I 0.10-0.12. Initial-velocity and product-inhibition results are consistent with an ordered sequential mechanism in which carbamoyl phosphate is the first substrate to bind, followed by L-aspartate, and carbamoyl aspartate is the first product to leave, followed by Pi. The order of substrate addition is supported by dead-end inhibition studies using pyrophosphate and maleate as inhibitory analogues of the substrates. Product inhibition permitted a minimum value for the dissociation constant of L-aspartate from the ternary complex to be estimated. This minimum is of the same order as the dissociation constant (Ki) of succinate. 2. A range of dicarboxy analogues of L-aspartate were tested as possible inhibitors of the enzyme. These studies suggested that L-aspartate is bound with its carboxy groups in the eclipsed configuration, and that the stereochemical constraints around the binding site are very similar to those reported for the catalytic subunit of the enzyme from Escherichia coli [Davies, Vanaman & Stark (1970) J. Biol. Chem. 245, 1175-1179].

Full text

PDF
247

Selected References

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

  1. CLELAND W. W. The kinetics of enzyme-catalyzed reactions with two or more substrates or products. I. Nomenclature and rate equations. Biochim Biophys Acta. 1963 Jan 8;67:104–137. doi: 10.1016/0006-3002(63)91800-6. [DOI] [PubMed] [Google Scholar]
  2. Chang T. Y., Jones M. E. Aspartate transcarbamylase from Streptococcus faecalis. Steady-state kinetic analysis. Biochemistry. 1974 Feb 12;13(4):638–645. doi: 10.1021/bi00701a002. [DOI] [PubMed] [Google Scholar]
  3. Cleland W. W. The statistical analysis of enzyme kinetic data. Adv Enzymol Relat Areas Mol Biol. 1967;29:1–32. doi: 10.1002/9780470122747.ch1. [DOI] [PubMed] [Google Scholar]
  4. Collins K. D., Stark G. R. Aspartate transcarbamylase. Studies of the catalytic subunit by ultraviolet difference spectroscopy. J Biol Chem. 1969 Apr 10;244(7):1869–1877. [PubMed] [Google Scholar]
  5. DIXON M. The determination of enzyme inhibitor constants. Biochem J. 1953 Aug;55(1):170–171. doi: 10.1042/bj0550170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Davies G. E., Vanaman T. C., Stark G. R. Aspartate transcarbamylase. Stereospecific restrictions on the binding site for L-aspartate. J Biol Chem. 1970 Mar 10;245(5):1175–1179. [PubMed] [Google Scholar]
  7. Grayson J. E., Yon R. J., Butterworth P. J. Wheat-germ aspartate transcarbamoylase. Purification and cold-lability. Biochem J. 1979 Nov 1;183(2):239–245. doi: 10.1042/bj1830239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Grayson J. E., Yon R. J. Wheat-germ aspartate transcarbamylase: reversible ligand-dependent aggregation behaviour in vitro. Biochem Soc Trans. 1978;6(1):197–200. doi: 10.1042/bst0060197. [DOI] [PubMed] [Google Scholar]
  9. Heyde E., Nagabhushanam A., Morrison J. F. Mechanism of the reaction catalyzed by the catalytic subunit of aspartate transcarbamylase. Kinetic studies with carbamyl phosphate as substrate. Biochemistry. 1973 Nov 6;12(23):4718–4726. doi: 10.1021/bi00747a600. [DOI] [PubMed] [Google Scholar]
  10. Jacobson G. R., Stark G. R. Aspartate transcarbamylase of Escherichia coli. Mechanisms of inhibition and activation by dicarboxylic acids and other anions. J Biol Chem. 1975 Sep 10;250(17):6852–6860. [PubMed] [Google Scholar]
  11. Lue P. F., Aitken D. M., Kaplan J. G. Studies of the regulation and reaction mechanism of the carbamyl phosphate synthetase and aspartate transcarbamylase of bakers' yeast. Biochimie. 1976;58(1-2):19–25. doi: 10.1016/s0300-9084(76)80352-5. [DOI] [PubMed] [Google Scholar]
  12. Ong B. L., Jackson J. F. Aspartate transcarbamoylase from Phaseolus aureus. Partial purification and properties. Biochem J. 1972 Sep;129(3):571–581. doi: 10.1042/bj1290571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Porter R. W., Modebe M. O., Stark G. R. Aspartate transcarbamylase. Kinetic studies of the catalytic subunit. J Biol Chem. 1969 Apr 10;244(7):1846–1859. [PubMed] [Google Scholar]
  14. Prescott L. M., Jones M. E. Modified methods for the determination of carbamyl aspartate. Anal Biochem. 1969 Dec;32(3):408–419. doi: 10.1016/s0003-2697(69)80008-4. [DOI] [PubMed] [Google Scholar]
  15. Wedler F. C., Gasser F. J. Ordered substrate binding and evidence for a thermally induced change in mechanism for E. coli aspartate transcarbamylase. Arch Biochem Biophys. 1974 Jul;163(1):57–68. doi: 10.1016/0003-9861(74)90454-8. [DOI] [PubMed] [Google Scholar]
  16. Yon R. J. Wheat-germ aspartate transcarbamoylase. Kinetic behaviour suggesting an allosteric mechanism of regulation. Biochem J. 1972 Jun;128(2):311–320. doi: 10.1042/bj1280311. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES