A single mutation in the regulatory chain of Escherichia coli aspartate transcarbamoylase results in an extreme T-state structure - PubMed

Affiliations

• PMID: 9680480
• DOI: 10.1006/jmbi.1998.1923

Comparative Study

A single mutation in the regulatory chain of Escherichia coli aspartate transcarbamoylase results in an extreme T-state structure

M K Williams et al. J Mol Biol. 1998.

Abstract

Kinetic analysis of a mutant version of Escherichia coli aspartate transcarbamoylase in which Thr82 in the regulatory chain (Thr82r) was replaced by Ala results in a shift in the T <==> R equilibrium towards the T-state. In order to understand the structural determinants of this T-state stabilization, the X-ray structure of the unliganded Thr82r-->Ala enzyme was determined at 2. 6 A resolution and refined to a crystallographic residual of 0.175. The structure of the mutant r1 regulatory chain is more similar to that of the r6 regulatory chain than observed for the wild-type enzyme, resulting in a more symmetric structure. Furthermore, the structural changes in the mutant enzyme appears to occur only in the r1 chain, while the r6 chain is almost identical in structure to that of the r6 chain of the wild-type enzyme. The structure of the mutant enzyme exhibits alterations in the subunit interfaces between the regulatory and catalytic chains, as well as in the interface between the allosteric and zinc domains within the regulatory chain. Moreover, the regulatory dimers are rotated around their respective 2-fold axes approximately 1 degrees beyond the rotation which occurs in the wild-type T-state enzyme. The structural analysis indicates that the enzyme is an "extreme" T-state, in which a larger rotation of the regulatory dimers is required for the T to R transition compared to the wild-type enzyme. This extreme T-state structure correlates well with the kinetic parameters determined for the mutant enzyme, showing a stabilized T-state. Furthermore, the structural analysis of the mutant enzyme suggests that replacement of Thr82r with Ala alters the local conformation of the nucleotide binding pocket and therefore offers a plausible explanation for the reduced affinity of the enzyme for nucleotides.

Similar articles

• Comparison of two T-state structures of regulatory-chain mutants of Escherichia coli aspartate transcarbamoylase suggests that His20 and Asp19 modulate the response to heterotropic effectors.

  Stec B, Williams MK, Stieglitz KA, Kantrowitz ER. Stec B, et al. Acta Crystallogr D Biol Crystallogr. 2007 Dec;63(Pt 12):1243-53. doi: 10.1107/S0907444907052985. Epub 2007 Nov 16. Acta Crystallogr D Biol Crystallogr. 2007. PMID: 18084072

• Modelling allosteric processes in E coli aspartate transcarbamylase.

  Cherfils J, Vachette P, Janin J. Cherfils J, et al. Biochimie. 1990 Aug;72(8):617-24. doi: 10.1016/0300-9084(90)90125-z. Biochimie. 1990. PMID: 2126466 Review.

• Structure and mechanisms of Escherichia coli aspartate transcarbamoylase.

  Lipscomb WN, Kantrowitz ER. Lipscomb WN, et al. Acc Chem Res. 2012 Mar 20;45(3):444-53. doi: 10.1021/ar200166p. Epub 2011 Oct 19. Acc Chem Res. 2012. PMID: 22011033 Free PMC article. Review.

Cited by

• From Genome to Structure and Back Again: A Family Portrait of the Transcarbamylases.

  Shi D, Allewell NM, Tuchman M. Shi D, et al. Int J Mol Sci. 2015 Aug 12;16(8):18836-64. doi: 10.3390/ijms160818836. Int J Mol Sci. 2015. PMID: 26274952 Free PMC article. Review.

• The use of nucleotide analogs to evaluate the mechanism of the heterotropic response of Escherichia coli aspartate transcarbamoylase.

  Sakash JB, Tsen A, Kantrowitz ER. Sakash JB, et al. Protein Sci. 2000 Jan;9(1):53-63. doi: 10.1110/ps.9.1.53. Protein Sci. 2000. PMID: 10739247 Free PMC article.

• Allosteric regulation of catalytic activity: Escherichia coli aspartate transcarbamoylase versus yeast chorismate mutase.

  Helmstaedt K, Krappmann S, Braus GH. Helmstaedt K, et al. Microbiol Mol Biol Rev. 2001 Sep;65(3):404-21, table of contents. doi: 10.1128/MMBR.65.3.404-421.2001. Microbiol Mol Biol Rev. 2001. PMID: 11528003 Free PMC article. Review.

• Structural basis for ordered substrate binding and cooperativity in aspartate transcarbamoylase.

  Wang J, Stieglitz KA, Cardia JP, Kantrowitz ER. Wang J, et al. Proc Natl Acad Sci U S A. 2005 Jun 21;102(25):8881-6. doi: 10.1073/pnas.0503742102. Epub 2005 Jun 10. Proc Natl Acad Sci U S A. 2005. PMID: 15951418 Free PMC article.

• Structure of the catalytic trimer of Methanococcus jannaschii aspartate transcarbamoylase in an orthorhombic crystal form.

  Vitali J, Colaneri MJ. Vitali J, et al. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008 Sep 1;64(Pt 9):776-80. doi: 10.1107/S1744309108025359. Epub 2008 Aug 20. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008. PMID: 18765902 Free PMC article.

Publication types

MeSH terms

Substances

LinkOut - more resources

• Full Text Sources

  • Elsevier Science

• Molecular Biology Databases

  • Saccharomyces Genome Database