The pyridoxal 5'-phosphate-dependent tryptophan synthase alpha(2)beta(2) 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 beta Ser-178 of beta helix 6 and the NH group of alpha Gly-181 of alpha loop 6. We investigated whether and to what extent mutations of alpha Gly-181 and beta Ser-178 affect allosteric regulation by the replacement of beta Ser-178 with Pro or Ala and of alpha Gly-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 beta Ser-178 and alpha Gly-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.

Identification of the Geometric Requirements for the Allosteric Communication between Alpha and Beta Subunits of Tryptophan Synthase / RABONI S.; BETTATI S.; MOZZARELLI A.. - In: THE JOURNAL OF BIOLOGICAL CHEMISTRY. - ISSN 0021-9258. - 280:14(2005), pp. 13450-13456. [10.1074/jbc.M414521200]

Identification of the Geometric Requirements for the Allosteric Communication between Alpha and Beta Subunits of Tryptophan Synthase

RABONI, Samanta;BETTATI, Stefano;MOZZARELLI, Andrea
2005

Abstract

The pyridoxal 5'-phosphate-dependent tryptophan synthase alpha(2)beta(2) 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 beta Ser-178 of beta helix 6 and the NH group of alpha Gly-181 of alpha loop 6. We investigated whether and to what extent mutations of alpha Gly-181 and beta Ser-178 affect allosteric regulation by the replacement of beta Ser-178 with Pro or Ala and of alpha Gly-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 beta Ser-178 and alpha Gly-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.
Identification of the Geometric Requirements for the Allosteric Communication between Alpha and Beta Subunits of Tryptophan Synthase / RABONI S.; BETTATI S.; MOZZARELLI A.. - In: THE JOURNAL OF BIOLOGICAL CHEMISTRY. - ISSN 0021-9258. - 280:14(2005), pp. 13450-13456. [10.1074/jbc.M414521200]
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11381/1444148
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