Characterization of a slow folding reaction for the alpha subunit of tryptophan synthase.
The equilibria and kinetics of urea-induced unfolding and refolding of the alpha subunit of tryptophan synthase of E. coli have been examined for their dependences on viscosity, pH, and temperature in order to investigate the properties of one of the rate-limiting steps, domain association. A viscosity enhancer, 0.58 M sucrose, was found to slow unfolding and accelerate refolding. This apparently anomalous result was shown to be due to the stabilizing effect of sucrose on the folding reaction. After accounting for this stabilization effect by using linear free-energy plots, the unfolding and refolding kinetics were found to have a viscosity dependence. A decrease in pH was found to stabilize the domain association reaction by increasing the refolding rate and decreasing the unfolding rate. This effect was accounted for by protonation of a single residue with a pK value of 8.8 in the native state and 7.1 in the intermediate, in which the two domains are not yet associated. The activation energy of unfolding is 4.8 kcal/mol, close to the diffusion limit. The negative activation entropy of unfolding, -47 cal/deg-mol, which controls this reaction, may result from ordering of solvent about the newly exposed domain interface of the transition state. These results may provide information on the types of noncovalent interactions involved in domain association and improve the ability to interpret the folding of mutants with single amino-acid substitutions at the interface.
Hurle
M R
MR
Department of Chemistry, Pennsylvania State University, University Park 16802.
Michelotti
G A
GA
Crisanti
M M
MM
Matthews
C R
CR
D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D008962:Models, Theoretical; D009154:Mutation; D011487:Protein Conformation / Q000187:drug effects; D013816:Thermodynamics; D014367:Tryptophan Synthase / Q000235:genetics; D014508:Urea / Q000494:pharmacology; D014783:Viscosity
D014508:Urea; D014367:Tryptophan Synthase