Thermal Repair of Tryptophan Synthase Mutations in a Regulatory Intersubunit Salt Bridge

This work is aimed at understanding how protein structure and conformation regulate activity and allosteric communication in the tryptophan synthase α2β2 complex from Salmonella typhimurium. Previous crystallographic and kinetic results suggest that both monovalent cations and a salt bridge between α subunit Asp56 and β subunit Lys167 play allosteric roles. Here we show that mutation of either of these salt bridging residues produced deleterious effects that could be repaired by increased temperature in combination with CsCl or with NaCl plus an α subunit ligand, α-glycerol 3-phosphate. Arrhenius plots of the activity data under these conditions were nonlinear. The same conditions yielded temperature-dependent changes in the equilibrium distribution of enzyme-substrate intermediates and in primary kinetic isotope effects. We correlate the results with a model in which the mutant enzymes are converted by increased temperature from a low activity, “open” conformation to a high activity, “closed” conformation under certain conditions. The allosteric ligand and different monovalent cations affected the equilibrium between the open and closed forms. The results suggest that α subunit Asp56 and β subunit Lys167 are not essential for catalysis and for allosteric communication between the α and β subunits but that their mutual interaction is important in stabilization of the active, closed form of the α2β2 complex.