Subunit structure and hybrid formation of bovine pyruvate kinases.
After denaturing either type M or L pyruvate kinase by guanidine hydrochloride, urea, or low pH, enzymatic activity and quaternary structure can be recovered by diluting the enzyme into buffer containing beta-mercaptoethanol. After denaturation of type M pyruvate kinase by guanidine hydrochloride, the yield and polarization of the intrinsic protein fluorescence, as well as most of the circular dichroism characteristic of the native enzyme, were regained very rapidly, while enzymatic activity was recovered much more slowly. Under the conditions used, about 50% of the original M and 30-50% of the original type L activity were typically recovered. Average half-times for recovery of enzymatic activity were 37 min for type M and 104 min for type L but depended somewhat on the renaturation buffer and on protein concentrations in the renaturation medium. If types M and L pyruvate kinases are renatured together, an approximately random recombination of the two subunits types results in a five-membered hybrid set. We have used this hybridizability to determine the kinetics of reformation of the native tetramer by denaturing each isozyme and beginning its renaturation separately at various times mixing the two isozymes and continuing their renaturation together. These studies indicate that reformation of stable tetramers occurs relatively slowly, qualitatively paralleling the regain of enzymatic activity, and that tetramer formation may be necessary for enzymatic activity. Using a similar technique to test for spontaneous dissociation of the native isozymes in buffer, we find that type L, but not type M, reversibly dissociates into dimers and monomers in buffer solutions. This dissociation is decreased by the presence of the substrate, phosphoenolpyruvate, by Mg2+ ions, or by the allosteric effector, fructose bisphosphate.
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D005636:Fructosephosphates / Q000494:pharmacology; D006824:Hybridization, Genetic; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008274:Magnesium / Q000494:pharmacology; D009132:Muscles / Q000201:enzymology; D010728:Phosphoenolpyruvate / Q000494:pharmacology; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D011770:Pyruvate Kinase / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013395:Sucrose / Q000494:pharmacology
D005636:Fructosephosphates; D007527:Isoenzymes; D013395:Sucrose; D010728:Phosphoenolpyruvate; D011770:Pyruvate Kinase; D008274:Magnesium