Triosephosphate isomerase (TIM), whose structure is archetypal of dimeric (beta/alpha)(8) barrels, has a conserved salt bridge (Arg189-Asp225 in yeast TIM) that connects the two C-terminal beta/alpha segments to rest of the monomer. We constructed the mutant D225Q, and studied its catalysis and stability in comparison with those of the wild-type enzyme. Replacement of Asp225 by Gln caused minor drops in k(cat) and K(M), but the catalytic efficiency (k(cat)/K(M)) was practically unaffected. Temperature-induced unfolding-refolding of both TIM samples displayed hysteresis cycles, indicative of processes far from equilibrium. Kinetic studies showed that the rate constant for unfolding was about three-fold larger in the mutant than in wild-type TIM. However, more drastic changes were found in the kinetics of refolding: upon mutation, the rate-limiting step changed from a second-order (at submicromolar concentrations) to a first-order reaction. These results thus indicate that renaturation of yTIM occurs through a uni-bimolecular mechanism in which refolding of the monomer most likely begins at the C-terminal half of its polypeptide chain. From the temperature dependence of the refolding rate, we determined the change in heat capacity for the formation of the transition state from unfolded monomers. The value for the D225Q mutant, which is about 40% of the corresponding value for yTIM, would implicate the folding of only three quarters of a monomer chain in the transition state. Proteins 2008. (c) 2008 Wiley-Liss, Inc.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Reference||Annotation Extension|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Conditions||Strain||Source||Reference|