Allosteric pyruvate kinase from Phycomyces blakesleeanus: physicochemical and regulatory properties

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Abstract
Pyryvate kinase from mycelium of Pycomyces blakesleeanus NRRL 1555 (-) was purified approximately 500-fold to a final specific activity of 25 U .cntdot. mg protein-1. The native enzyme has a molecular weight of 230 000. The enzyme appeared to be a tetramer with apparently identical subunits of 58 000 each. The enzyme from Phycomyces required Mg2+ for activity, but not K+ or NH4. It showed a transition temperature of 36.degree. C. L-Alanine and ATP allosterically inhibited the enzyme by increasing the positive homotropic interactions for phosphoenolpyruvate and abolishing them for Mg2+ ions. Both effectors appeared to be competitive inhibitors with regard to ADP. Fructose 1,6-bisphosphate activates the Phycomyces pyruvate kinase allosterically by transforming the sigmoidal saturation curces to a hyperbolic form for phosphoenolpyruvate and Mg2+. Furthermore, fructose 1,6-bisphosphate relieved the inhibition caused by ATP and L-alanine. A lowering of the pH for reaction also activates the enzyme by abolishing the sigmoidal saturation curve for phosphoenolpyruvate, but produces substrate inhibition. The kinetic properties of Phycomyces pyruvate kinase are compatible to that of an allosteric K-type enzyme.