title: Thermal repair of tryptophan synthase mutations in a regulatory intersubunit salt bridge. Evidence from arrhenius plots, absorption spectra, and primary kinetic isotope effects.
abstract: This work is aimed at understanding how protein structure and conformation regulate activity and allosteric communication in the tryptophan synthase alpha(2)beta(2) complex from Salmonella typhimurium. Previous crystallographic and kinetic results suggest that both monovalent cations and a salt bridge 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 temperature in combination with CsCl or with NaCl plus an alpha subunit ligand, alpha-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 alpha subunit Asp(56) and beta subunit Lys(167) are not essential for catalysis and for allosteric communication between the alpha and beta subunits but that their mutual interaction is important in stabilization of the active, closed form of the alpha(2)beta(2) complex.
authors.affiliation: Section on Enzyme Structure and Function, Laboratory of Biochemistry and Genetics, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-0830, USA.
authors.forename: Y X
authors.initials: YX
authors.lastname: Fan
authors.forename: P
authors.initials: P
authors.lastname: McPhie
authors.forename: E W
authors.initials: EW
authors.lastname: Miles
mesh_terms: D000494:Allosteric Regulation; D001426:Bacterial Proteins / Q000235:genetics; D002414:Cations, Monovalent / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005994:Glycerophosphates / Q000494:pharmacology; D007211:Indoles / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D011487:Protein Conformation; D012486:Salmonella typhimurium / Q000201:enzymology*; D012694:Serine / Q000378:metabolism; D013053:Spectrophotometry; D013696:Temperature; D013816:Thermodynamics; D014367:Tryptophan Synthase / Q000737:chemistry* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D002414:Cations, Monovalent; D005994:Glycerophosphates; D007211:Indoles; D012694:Serine; C030374:indole; C029620:alpha-glycerophosphoric acid; D014367:Tryptophan Synthase