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.
Highlights
Regulation of enzyme activity by interaction with metabolites is a key feature for the maintenance of cell life
The comparison of the three-dimensional structure of tryptophan synthase (TS) in the absence and presence of a ligand in the ␣-active site and the catalytic intermediate ␣aminoacrylate in the -active site [5, 15] evidenced several structural differences: (i) the formation of hydrogen bonds between ␣ loop 2 and  helix 6, (ii) the stabilization of ␣ loop 6, which is crystallographically undetectable in the open form due to high mobility [5, 10], in a closed form (Fig. 1), (iii) the presence of a single hydrogen bond between ␣ loop 6 and  helix 6, and (iv) the movement of part of the -subunit, including  helix 6
These data indicate that the ligandmediated intersubunit communication has been interrupted, suggesting that mutations do not allow the formation of the hydrogen bond between positions 178 and ␣181
Summary
Regulation of enzyme activity by interaction with metabolites is a key feature for the maintenance of cell life. The comparison of the three-dimensional structure of TS in the absence (both ␣- and -subunits in the open state) and presence of a ligand in the ␣-active site and the catalytic intermediate ␣aminoacrylate in the -active site (both ␣- and -subunits in the closed state) [5, 15] evidenced several structural differences: (i) the formation of hydrogen bonds between ␣ loop 2 and  helix 6, (ii) the stabilization of ␣ loop 6, which is crystallographically undetectable in the open form due to high mobility [5, 10], in a closed form (Fig. 1), (iii) the presence of a single hydrogen bond between ␣ loop 6 and  helix 6, and (iv) the movement of part of the -subunit, including  helix 6 This mobile portion of the -subunit was called the COMM domain because of its role in communicating to the -active site conformational changes occurring at the ␣-active site. By taking into account that ␣Gly-181, but not Ser-178, is a conserved residue among TS from several species [7, 32], we have investigated the geometric requirements for the achievement of the Ser-178-␣Gly-181 hydrogen bond by preparing ␣G181A, ␣G181V, and ␣G181F, mutants characterized by a progressively increasing side chain steric hindrance, and ␣G181P
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