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Identification of the geometric requirements for allosteric communication between the alpha- and beta-subunits of tryptophan synthase.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2005 Apr 08; Vol. 280 (14), pp. 13450-6. Date of Electronic Publication: 2005 Feb 03. - Publication Year :
- 2005
-
Abstract
- The pyridoxal 5'-phosphate-dependent tryptophan synthase alpha2beta2 complex is a paradigmatic protein for substrate channeling and allosteric regulation. The enzymatic activity is modulated by a ligand-mediated equilibrium between open (inactive) and closed (active) conformations of the alpha- and beta-subunit, predominantly involving the mobile alpha loop 6 and the beta-COMM domain that contains beta helix 6. The alpha ligand-triggered intersubunit communication seems to rely on a single hydrogen bond formed between the carbonyl oxygen of betaSer-178 of beta helix 6 and the NH group of alphaGly-181 of alpha loop 6. We investigated whether and to what extent mutations of alphaGly-181 and betaSer-178 affect allosteric regulation by the replacement of betaSer-178 with Pro or Ala and of alphaGly-181 with either Pro to remove the amidic proton that forms the hydrogen bond or Ala, Val, and Phe to analyze the dependence on steric hindrance of the open-closed conformational transition. The alpha and beta activity assays and the equilibrium distribution of beta-subunit catalytic intermediates indicate that mutations do not significantly influence the intersubunit catalytic activation but completely abolish ligand-induced alpha-to beta-subunit signaling, demonstrating distinct pathways for alpha-beta-site communication. Limited proteolysis experiments indicate that the removal of the interaction between betaSer-178 and alphaGly-181 strongly favors the more trypsin-accessible open conformation of the alpha-active site. When the hydrogen bond cannot be formed, the alpha-subunit is unable to attain the closed conformation, and consequently, the allosteric signal is aborted at the subunit interface.
- Subjects :
- Allosteric Regulation
Bacterial Proteins chemistry
Bacterial Proteins metabolism
Binding Sites
Models, Molecular
Molecular Structure
Mutagenesis, Site-Directed
Salmonella typhimurium enzymology
Serine metabolism
Protein Conformation
Protein Subunits chemistry
Protein Subunits metabolism
Tryptophan Synthase chemistry
Tryptophan Synthase metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 280
- Issue :
- 14
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 15691828
- Full Text :
- https://doi.org/10.1074/jbc.M414521200