Purification and Allosteric Properities of Yeast Pyruvate Kinase

Abstract

Yeast pyruvate kinase was purified approximately 100-fold by a procedure involving cytolysis by toluene, alkaline extraction, fractionation with ammonium sulphate, adsorption on Blue Dextran and repetitive gelfiltrations. The specific activity of the stable enzyme was 200 [mu]moles [center dot] min-1.mg enzyme protein-1, for which a molecular weight of 200,000 was determined by gel filtration. Allosteric activation was observed with the following ligands: phospho-enolpyruvate (nfl = 2.6), ADP (nH = 1.4), Fru-1,6-Po (nH = 2.7), Mg2 (rip = 2.7),Rb+(Hjj=Z.7). Low concentrations of ATP and Ca2+excreted. activation under specified conditions. Allosteric inhibition was observed with ATP (nH>5), citrate (nH = 3), NADP (iur = 6.3), Ca2 (nH = 2.1). Inhibitions were also observed with CTP, GTP, UTP, ITP, AMP and cyclic AMP. Fructose, 1,6-diphosphate (Fru-l^-P2,) interferes with the activation by monovalent ions and markedly decreases the Kjj^-value for phosphoenolpyruvate, transforming the sigmoidal phospno-enolpyruvate characteristic to a hyperbolic curvature. Fru-1,6-P2 also abolishes an ionized group of the enzyme-phosphoenolpyruvate-com-plex with a pK-value of 7.0. The transformation of the enzyme by Fru-1,6-P2,, can also be detected by its loss of binding sites for Blue Dextran. Hexitoldiphosphate was found to be a strong inhibitor of the enzyme. The data are discussed in terms of the current allosteric theories of enzyme action as well as with respect to its significance for the operation of pyruvate kinase as a control unit in glycolysis.