Analysis of the renaturation kinetics of bovine muscle pyruvate kinase

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Abstract
Bovine type M pyruvate kinase can be reversibly denatured by solutions of guanidine-hydrochloride. Subsequent dilution of the enzyme into buffer containing 2-mercaptoethanol or dithiothreitol results in recovery of enzymatic activity with half-times that vary from 185 min at 0.degree. C to 4 min at 45.degree. C. In the temperature range 0-25.degree. C, 90% of the enzymatic activity is recovered. Above about 32.degree. C, the recovery drops off sharply, with a yield of only 13% at 45.degree. C. Removal of inactive nonspecific aggregates and denatured monomer by gel filtration yields an enzyme with the same specific activity as the starting material. At enzyme concentrations below 3 .mu.g/ml at 16.degree. C or below 25 .mu.g/ml at 7.8.degree. C, the reactivation kinetics show a concentration dependence. At higher concentrations of protein and at temperatures of 16.degree. C or higher, no protein concentration dependence is seen, and the rate of reactivation is described by 2 first-order relaxations. The rate constants have apparent activation energies of 10.6 and 11.9 kcal/mol. A rapid, major folding may produce 2 species which undergo transconformational steps. This is followed by subunit association which yields the native tetramer.