The reconstitution of denatured phosphoglycerate mutase

The reconstitution of the tetrameric enzyme yeast phosphoglycerate mutase after denaturation in guanidine hydrochloride has been studied. Denaturation is almost completely reversible at enzyme concentrations greater than 10 micrograms/ml. Cross-linking by glutaraldehyde has been used to monitor the reassociation of the subunits; the kinetics of this process has been analyzed in terms of a model involving an equilibrium between monomer and dimer followed by a bimolecular association of two dimers to give a tetramer. Reactivation is found to parallel the appearance of tetramer. Structural changes during reconstitution have been measured by circular dichroism and fluorescence. Both methods reveal complex kinetics indicating the rapid formation of structured monomers (half-time less than 10 s), followed by slow subunit association. For comparison, preliminary reconstitution experiments were performed on the dimeric phosphoglycerate mutase from rabbit muscle.