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J. Biol. Chem., Vol. 276, Issue 21, 17747-17753, May 25, 2001

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Allosteric Communication of Tryptophan Synthase
FUNCTIONAL AND REGULATORY PROPERTIES OF THE S178P MUTANT^*

Anna Marabotti, Daniela De Biase^§, Angela Tramonti^§, Stefano Bettati^¶, and Andrea Mozzarelli^¶

From the  Institute of Biochemical Sciences and ^¶ National Institute for the Physics of Matter, University of Parma, 43100 Parma, Italy and the ^§ Department of Biochemical Sciences "A. Rossi Fanelli," University of Rome "La Sapienza," 00185 Rome, Italy

The ₂₂ tryptophan synthase complex is a model enzyme for understanding allosteric regulation. We report the functional and regulatory properties of the S178P mutant. Ser-178 is located at the end of helix 6 of the  subunit, belonging to the domain involved in intersubunit signaling. The carbonyl group of Ser-178 is hydrogen bonded to Gly-181 of loop 6 of the  subunit only when  subunit ligands are bound. An analysis by molecular modeling of the structural effects caused by the S178P mutation suggests that the hydrogen bond involving Gly-181 is disrupted as a result of localized structural perturbations. The ratio of  to  subunit concentrations was calculated to be 0.7, as for the wild type, indicating the maintenance of a tight complex. Both the activity of the  subunit and the inhibitory effect of the  subunit ligands indole-3-acetylglycine and D,L--glycerol-3-phosphate were found to be the same for the mutant and wild type enzyme, whereas the  subunit activity of the mutant exhibited a 2-fold decrease. In striking contrast to that observed for the wild type, the allosteric effectors indole-3-acetylglycine and D,L--glycerol-3-phosphate do not affect the  activity. Accordingly, the distribution of L-serine intermediates at the -site, dominated by the -aminoacrylate, is only slightly influenced by  subunit ligands. Binding of sodium ions is weaker in the mutant than in the wild type and leads to a limited increase of the amount of the external aldimine intermediate, even at high pH, whereas binding of cesium ions exhibits the same affinity and effects as in the wild type, leading to an increase of the -aminoacrylate tautomer absorbing at 450 nm. Crystals of the S178P mutant were grown, and their functional and regulatory properties were investigated by polarized absorption microspectrophotometry. These findings indicate that (i) the reciprocal activation of the  and  activity in the 22 complex with respect to the isolated subunits results from interactions that involve residues different from Ser-178 and (ii) Ser-178 is a critical residue in ligand-triggered signals between  and  active sites.

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