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

Allosteric communications are important in coordination of the reactions in the tryptophan (Trp) synthase α2β2 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 (Na2GP), indoleacetylglycine (IAG), and benzimidazole (BZI), as well as of βE109D and βD305A mutations, on Keq for the conformational equilibria were determined. The l-Ser external aldimine–aminoacrylate equilibrium (Keq = [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), NH4+ (2.3 × 10−4), no cation (2.0 × 10−4) and Cs+ (1.6 × 10−5). α-Site ligands, Na2GP and IAG, have modest 3- to 40-fold effects on Keq in the direction of aminoacrylate, but BZI in the presence of Na+ gives a low value of Keq comparable to that obtained with Cs+. There is no additivity of free energy for Na2GP and BZI, suggesting a common pathway for allosteric communications for both ligands. The values of ΔVo range from −126 mL/mol for the Na+ complex to −204 mL/mol for the Na+ complex with BZI. The βD305A mutation changes the Keq by a factor of at least 105 (26.7 kJ/mol) and nearly abolishes allosteric communications. There are also dramatic decreases in the magnitude of both ΔVo and ΔS 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 Keq, 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.