Thermal repair of tryptophan synthase mutations in a regulatory intersubunit salt bridge - Evidence from arrhenius plots, absorption spectra, and primary kinetic isotope effects

This work is aimed at understanding how protein structure and conformation regulate activity and allosteric communication in the tryptophan synthase a lpha(2)beta(2) complex from Salmonella typhimurium. Previous erystallograph ic and kinetic results suggest that both monovalent cations and a salt brid ge between alpha subunit Asp(56) and beta subunit Lys(167) play allosteric roles. Here we show that mutation of either of these salt bridging residues produced deleterious effects that could be repaired by increased temperatu re in combination with CsCl or with NaCl plus an alpha subunit ligand, alph a-glycerol 3-phosphate. Arrhenius plots of the activity data under these co nditions were nonlinear, The same conditions yielded temperature-dependent changes in the equilibrium distribution of enzyme-substrate intermediates a nd in primary kinetic isotope effects. We correlate the results with a mode l in which the mutant enzymes are converted by increased temperature from a low activity, "open" conformation to a high activity, "closed" conformatio n under certain conditions. The allosteric ligand and different monovalent cations affected the equilibrium between the open and closed forms. The res ults suggest that alpha subunit Asp(56) and beta subunit Lys(167) are not e ssential for catalysis and for allosteric communication between the alpha a nd beta subunits but that their mutual interaction is important in stabiliz ation of the active, closed form of the alpha(2)beta(2) complex.