Allosteric communication of tryptophan synthase - Functional and regulatory properties of the beta S178P mutant

The alpha (2)beta (2) tryptophan synthase complex is a model enzyme for und erstanding allosteric regulation. We report the functional and regulatory p roperties of the beta S178P mutant. Ser-178 is located at the end of helix 6 of the beta subunit, belonging to the domain involved in intersubunit sig naling. The carbonyl group of beta Ser-178 is hydrogen bonded to Gly-181 of loop 6 of the alpha subunit only when alpha subunit ligands are bound. An analysis by molecular modeling of the structural effects caused by the beta S178P mutation suggests that the hydrogen bond involving alpha Gly-181 is disrupted as a result of localized structural perturbations. The ratio of a lpha to beta subunit concentrations was calculated to be 0.7, as for the wi ld type, indicating the maintenance of a tight alpha-beta complex. Both the activity of the alpha subunit and the inhibitory effect of the alpha subun it ligands indole-3-acetylglycine and D,L-alpha -glycerol-3-phosphate were found to be the same for the mutant and wild type enzyme, whereas the beta subunit activity of the mutant exhibited a 2-fold decrease. In striking con trast to that observed for the wild type, the allosteric effecters indole-3 -acetylglycine and D,L-alpha -glycerol-3-phosphate do not affect the beta a ctivity. Accordingly, the distribution of L-serine intermediates at the bet a -site, dominated by the alpha -aminoacrylate, is only slightly influenced by alpha subunit ligands. Binding of sodium ions is weaker in the mutant t han in the wild type and leads to a limited increase of the amount of the e xternal aldimine intermediate, even at high pH, whereas binding of cesium i ons exhibits the same affinity and effects as in the wild type, leading to an increase of the alpha -aminoacrylate tautomer absorbing at 450 nm, Cryst als of the beta S178P mutant were grown, and their functional and regulator y properties were investigated by polarized absorption microspectrophotomet ry. These findings indicate that (i) the reciprocal activation of the alpha and beta activity in the alpha2 beta2 complex with respect to the isolated subunits results from interactions that involve residues different from be ta Ser-178 and (ii) beta Ser178 is a critical residue in ligand-triggered s ignals between alpha and beta active sites.