The pyridoxal 5-phosphate-dependent tryptophan synthase ␣ 2 ␤ 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 ␣and ␤-subunit, predominantly involving the mobile ␣ loop 6 and the ␤-COMM domain that contains ␤ helix 6. The ␣ ligand-triggered intersubunit communication seems to rely on a single hydrogen bond formed between the

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... carbonyl oxygen of ␤Ser-178 of ␤ helix 6 and the NH group of ␣Gly-181 of ␣ loop 6. We investigated whether and to what extent mutations of ␣Gly-181 and ␤Ser-178 affect allosteric regulation by the replacement of ␤Ser-178 with Pro or Ala and of ␣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 ␣ and ␤ activity assays and the equilibrium distribution of ␤-subunit catalytic intermediates indicate that mutations do not significantly influence the intersubunit catalytic activation but completely abolish ligand-induced ␣to ␤-subunit signaling, demonstrating distinct pathways for ␣-␤-site communication. Limited proteolysis experiments indicate that the removal of the interaction between ␤Ser-178 and ␣Gly-181 strongly favors the more trypsin-accessible open conformation of the ␣-active site. When the hydrogen bond cannot be formed, the ␣-subunit is unable to attain the closed conformation, and consequently, the allosteric signal is aborted at the subunit interface. Regulation of enzyme activity by interaction with metabolites is a key feature for the maintenance of cell life. This goal is achieved through conformational changes that, depending on protein complexity, involve tertiary and/or quaternary states. A common event that accompanies ligand binding to proteins is the transition between open and closed states (Ref. 1 and the references therein). Usually, the closed state is a more active conformation, bringing substrates and catalytic residues in close proximity, conferring special features to the active site environment, and protecting the productive catalysis from side reactions, generally involving water molecules.