Quantitative effects of allosteric ligands and mutations on conformational equilibria in Salmonella typhimurium tryptophan synthase

Abstract

Allosteric communications are important in coordination of the reactions in the tryptophan (Trp) synthase α 2β 2 multienzyme complex. We have measured the conformational equilibria of l-Ser and l-Trp complexes, using absorption and fluorescence spectrophotometry with hydrostatic pressure equilibrium perturbation. The effects of monovalent cations, disodium α-glycerophosphate (Na 2GP), indoleacetylglycine (IAG), and benzimidazole (BZI), as well as of βE109D and βD305A mutations, on K eq for the conformational equilibria were determined. The l-Ser external aldimine–aminoacrylate equilibrium ( K eq = [external aldimine]/[aminoacrylate]) has the largest value with Na + (0.12), followed by K + (0.04), Li + (7.6 × 10 −4), Rb + (4.3 × 10 −4), NH 4 + (2.3 × 10 −4), no cation (2.0 × 10 −4) and Cs + (1.6 × 10 −5). α-Site ligands, Na 2GP and IAG, have modest 3- to 40-fold effects on K eq in the direction of aminoacrylate, but BZI in the presence of Na + gives a low value of K eq comparable to that obtained with Cs +. There is no additivity of free energy for Na 2GP and BZI, suggesting a common pathway for allosteric communications for both ligands. The values of Δ V o range from −126 mL/mol for the Na + complex to −204 mL/mol for the Na + complex with BZI. The βD305A mutation changes the K eq by a factor of at least 10 5 (26.7 kJ/mol) and nearly abolishes allosteric communications. There are also dramatic decreases in the magnitude of both Δ V o and Δ S for the l-Ser external aldimine–aminoacrylate equilibrium for βD305A Trp synthase, consistent with a large decrease in solvation accompanying the conformational change in βD305A Trp synthase relative to wild-type Trp synthase. The βE109D mutation has more modest but significant effects on K eq, which differ with the ligand, ranging from 40-fold for GP to 2200-fold for BZI, even though βGlu-109 is not directly involved in allosteric communications. The effect of GP on the external aldimine–quinonoid intermediate equilibrium of the Trp synthase- l-Trp complex is similar to that of GP on the Trp synthase- l-Ser external aldimine–aminoacrylate equilibrium. These results have allowed a quantitative comparison of the allosteric effects of ligand and mutations in Trp synthase. These allosteric effects are finely tuned to control the synthesis of l-Trp without resulting in substrate or product inhibition.