A study of the allosteric kinetics of Phycomyces pyruvate kinase as judged by the effect of l-alanine and fructose 1,6-bisphosphate

Abstract

The influence of fructose 1,6-bisphosphate and l-alanine on the kinetics of pyruvate kinase (ATP: pyruvate O 2-phosphotransferase, EC 2.7.1.40) from Phycomyces blakesleeanus NRRL 1555 (−) was studied at pH 7.5. By addition of fructose 1,6-bisphosphate the sigmoid kinetics with respect to phospho enolpyruvate and Mg 2− were abolished and the velocity curves became hyperbolic. In the presence of l-alanine the positive homotropic cooperativity with respect to phospho enolpyruvate increased with Hill coefficient values close to 4, while the sigmoid kinetics with respect to Mg 2+ became hyperbolic. Fructose 1,6-bisphosphate overcomes the inhibition produced by l-alanine, the antagonism between phospho enolpyruvate and l-alanine also being evident. Inhibition has been found at high Mg 2+ concentrations, compatible with the binding of the magnesium ions to an inactive conformational state of the enzyme. The data were analysed on the basis of the two-states concerted-symmetry model of Monod, Wyman and Changeux, and the parameters of the model were calculated. Phospho enolpyruvate and fructose 1,6-bisphosphate appeared to show exclusive binding to the active conformational state (R), whereas magnesium ions bind preferentially, by a factor of 45, to the R state. l-Alanine binds more readily to the inactive T state of the enzyme.