Folding and association of triose phosphate isomerase from rabbit muscle.

The enzymatic activity and quaternary structure of rabbit muscle triose phosphate isomerase remains unchanged in the concentration range from 2 micrograms/ml to 2 ng/ml. In this concentration range the enzyme can be reactivated after dissociation and denaturation in 6.5 M guanidine hydrochloride. Removal of the denaturant by dilution and separation of inactive wrong aggregates (5-20%) lead back to active dimers, indistinguishable from the native enzymes as far as enzymatic and physicochemical properties are concerned. Based on the long term stability of the enzyme, the reactivation kinetics were analyzed at low concentrations and 0 degrees C, conditions where the association of inactive monomers to active dimers is predominant in the process of reactivation. The concentration dependence of the rate of reactivation and the kinetic profiles could be described by a consecutive first-order folding and second-order association reaction scheme with the rate constants kuni = 1.9 X 10(-2)s-1 and kbi = 3 X 10(5) M x s-1. This implies that the folded monomers of triose phosphate isomerase, which are intermediate states during reconstitution, cannot possess appreciable enzymatic activity.