Allosteric Communication of Tryptophan Synthase: FUNCTIONAL AND REGULATORY PROPERTIES OF THE βS178P MUTANT

Anna Marabotti,Daniela De Biase,Angela Tramonti,Stefano Bettati,Andrea Mozzarelli

doi:10.1074/jbc.m011781200

Abstract

The alpha(2)beta(2) tryptophan synthase complex is a model enzyme for understanding allosteric regulation. We report the functional and regulatory properties of the betaS178P mutant. Ser-178 is located at the end of helix 6 of the beta subunit, belonging to the domain involved in intersubunit signaling. The carbonyl group of betaSer-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 betaS178P mutation suggests that the hydrogen bond involving alphaGly-181 is disrupted as a result of localized structural perturbations. The ratio of alpha to beta subunit concentrations was calculated to be 0.7, as for the wild type, indicating the maintenance of a tight alpha-beta complex. Both the activity of the alpha subunit and the inhibitory effect of the alpha subunit 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 contrast to that observed for the wild type, the allosteric effectors indole-3-acetylglycine and d,l-alpha-glycerol-3-phosphate do not affect the beta activity. Accordingly, the distribution of l-serine intermediates at the beta-site, dominated by the alpha-aminoacrylate, is only slightly influenced by alpha 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 alpha-aminoacrylate tautomer absorbing at 450 nm. Crystals of the betaS178P 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 alpha and beta activity in the alpha2beta2 complex with respect to the isolated subunits results from interactions that involve residues different from betaSer-178 and (ii) betaSer-178 is a critical residue in ligand-triggered signals between alpha and beta active sites.

Highlights

The ␣2␤2 tryptophan synthase complex is a model enzyme for understanding allosteric regulation
Allosteric Regulation of Tryptophan Synthase proline was that the interaction between ␤Ser-178 and ␣Gly181 is mediated by a hydrogen bond between the carbonyl oxygen of ␤Ser-178 and the amidic nitrogen of ␣Gly-181. ␤Ser178 is not a conserved residue, as demonstrated by a multiple alignment of the amino acid sequence of ␤ subunits from different sources
A model of the structural pathway for the transmission of allosteric signals between ␣ and ␤ subunits of tryptophan synthase was proposed on the basis of fluorescence and phosphorescence properties of ␤Trp-177 in the absence and presence of ␣ subunit ligands [29, 30]

Summary

Introduction

The ␣2␤2 tryptophan synthase complex is a model enzyme for understanding allosteric regulation. 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. 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 ␣2␤2 complex with respect to the isolated subunits results from interactions that involve residues different from ␤Ser-178 and (ii) ␤Ser178 is a critical residue in ligand-triggered signals between ␣ and ␤ active sites. Allosteric Regulation of Tryptophan Synthase proline was that the interaction between ␤Ser-178 and ␣Gly181 is mediated by a hydrogen bond between the carbonyl oxygen of ␤Ser-178 and the amidic nitrogen of ␣Gly-181. ␤Ser178 is not a conserved residue, as demonstrated by a multiple alignment of the amino acid sequence of ␤ subunits from different sources. proline mutation was chosen for its unique feature of preventing the formation of the hydrogen bond with ␣Gly-181.3 to obtain structure and function relationships, crystals of the mutant were grown, and the catalytic competence of the mutant was investigated by polarized absorption microspectrophotometry

Methods

Results

Conclusion