Analysis of the renaturation kinetics of bovine muscle pyruvate kinase.

Bovine type M pyruvate kinase can be reversibly denatured by solutions of guanidine-HCl. 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 degrees C to 4 min at 45 degrees C. In the temperature range 0-25 degrees C, 90% of the enzymatic activity is recovered. Above about 32 degrees C, the recovery drops off sharply, wih a yield of only 13% at 45 degrees 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 microns/mL at 16 degrees C or below 25 micron/mL at 7.8 degrees C, the reactivation kinetics show a concentration dependence. At higher concentrations of protein and at temperatures of 16 degrees C or higher, no protein concentration dependence is seen, and the rate of reactivation is described by two first-order relaxations. The rate constants have apparent activation energies of 10.6 and 11.9 kcal/mol. Combinding the results presented here with earlier work from this laboratory [Cardenas, J. M., & Dyson, R. D. (1973) J. Biol. Chem. 248, 6938-6944; Cardenas, J. M., Hubbard, D. R., & Anderson, S. (1977) Biochemistry 16, 191-197] leads to the conclusion that a rapid, major folding produces two species which undergo transconformational steps. This is followed by subunit association which yields the native tetramer.