Skip to main content
PMC 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
. 1980 Jun;77(6):3249–3253. doi: 10.1073/pnas.77.6.3249

Isolation and preliminary characterization of single amino acid substitution mutants of aspartate carbamoyltransferase.

E R Kantrowitz, J Foote, H W Reed, L A Vensel
PMCID: PMC349592  PMID: 6997873

Abstract

In order to isolate functional Escherichia coli aspartate carbamoyltransferase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC2.1.3.2) with single amino acid replacements, a series of pyrB nonsense mutants has been isolated. These nonsense mutants were induced by 2-aminopurine mutagenesis and selected by a combination of antibiotic treatments, direct enzyme assays, and suppressibility tests. Suppression of the pyrB nonsense mutation with various suppressors, which insert different amino acids, has resulted in the formation of a series of mutant aspartate carbamoyltransferases, each differing in one amino acid from the wild-type enzyme. After partial purification, kinetic studies revealed that some of the mutant enzymes had altered homotropic and heterotropic interactions. The mutants that had a tyrosine insert showed the most pronounced changes, followed by those with a serine insert. The mutants having a glutamine insert, howevr, were indistinguishable from the wild-type enzyme, supporting the conclusion that, because of the specificity of the mutagen, the glutamine insert had regenerated the wild-type enzyme.

Full text

PDF
3249

Selected References

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

  1. Bachmann B. J., Low K. B., Taylor A. L. Recalibrated linkage map of Escherichia coli K-12. Bacteriol Rev. 1976 Mar;40(1):116–167. doi: 10.1128/br.40.1.116-167.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bethell M. R., Smith K. E., White J. S., Jones M. E. Carbamyl phosphate: an allosteric substrate for aspartate transcarbamylase of Escherichia coli. Proc Natl Acad Sci U S A. 1968 Aug;60(4):1442–1449. doi: 10.1073/pnas.60.4.1442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. GERHART J. C., PARDEE A. B. The enzymology of control by feedback inhibition. J Biol Chem. 1962 Mar;237:891–896. [PubMed] [Google Scholar]
  4. Gerhart J. C., Holoubek H. The purification of aspartate transcarbamylase of Escherichia coli and separation of its protein subunits. J Biol Chem. 1967 Jun 25;242(12):2886–2892. [PubMed] [Google Scholar]
  5. Kantrowitz E. R., Lipscomb W. N. An essential residue at the active site of aspartate transcarbamylase. J Biol Chem. 1976 May 10;251(9):2688–2695. [PubMed] [Google Scholar]
  6. Kolata G. B. Protein structure: systematic alteration of amino Acid sequences. Science. 1976 Jan 30;191(4225):373–373. doi: 10.1126/science.191.4225.373. [DOI] [PubMed] [Google Scholar]
  7. Low B. Rapid mapping of conditional and auxotrophic mutations in Escherichia coli K-12. J Bacteriol. 1973 Feb;113(2):798–812. doi: 10.1128/jb.113.2.798-812.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Meighen E. A., Pigiet V., Schachman H. K. Hybridization of native and chemically modified enzymes. 3. The catalytic subunits of aspartate transcarbamylase. Proc Natl Acad Sci U S A. 1970 Jan;65(1):234–241. doi: 10.1073/pnas.65.1.234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Monaco H. L., Crawford J. L., Lipscomb W. N. Three-dimensional structures of aspartate carbamoyltransferase from Escherichia coli and of its complex with cytidine triphosphate. Proc Natl Acad Sci U S A. 1978 Nov;75(11):5276–5280. doi: 10.1073/pnas.75.11.5276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. PARDEE A. B., YATES R. A. Control of pyrimidine biosynthesis in Escherichia coli by a feed-back mechanism. J Biol Chem. 1956 Aug;221(2):757–770. [PubMed] [Google Scholar]
  11. Pastra-Landis S. C., Evans D. R., Lipscomb W. N. The effect of pH on the cooperative behavior of aspartate transcarbamylase from Escherichia coli. J Biol Chem. 1978 Jul 10;253(13):4624–4630. [PubMed] [Google Scholar]
  12. Perbal B., Hervé G. Biosynthesis of Escherichia coli aspartate transcarbamylase. I. Parameters of gene expression and sequential biosynthesis of the subunits. J Mol Biol. 1972 Oct 14;70(3):511–529. doi: 10.1016/0022-2836(72)90556-6. [DOI] [PubMed] [Google Scholar]
  13. Perutz M. F., Lehmann H. Molecular pathology of human haemoglobin. Nature. 1968 Aug 31;219(5157):902–909. doi: 10.1038/219902a0. [DOI] [PubMed] [Google Scholar]
  14. Perutz M. F. Stereochemistry of cooperative effects in haemoglobin. Nature. 1970 Nov 21;228(5273):726–739. doi: 10.1038/228726a0. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Weber K. New structural model of E. coli aspartate transcarbamylase and the amino-acid sequence of the regulatory polypeptide chain. Nature. 1968 Jun 22;218(5147):1116–1119. doi: 10.1038/2181116a0. [DOI] [PubMed] [Google Scholar]
  17. Wiley D. C., Lipscomb W. N. Crystallographic determination of symmetry of aspartate transcarbamylase. Nature. 1968 Jun 22;218(5147):1119–1121. doi: 10.1038/2181119a0. [DOI] [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