A study of the allosteric kinetics of Phycomyces pyruvate kinase as judged by the effect of L-alanine and fructose 1,6-bisphosphate.
The influence of fructose 1,6-bisphosphate and L-alanine on the kinetics of pyruvate kinase (ATP:pyruvate O2-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 phosphoenol pyruvate and Mg2+ were abolished and the velocity curves became hyperbolic. In the presence of L-alanine the positive homotropic cooperativity with respect to phosphoenol pyruvate increased with Hill coefficient values close to 4, while the sigmoid kinetics with respect to Mg2+ became hyperbolic. Fructose 1,6-bisphosphate overcomes the inhibition produced by L-alanine, the antagonism between phosphoenol pyruvate and L-alanine also being evident. Inhibition has been found at high Mg2+ 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. Phosphoenol pyruvate 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.
D000409:Alanine / Q000494:pharmacology*; D000494:Allosteric Regulation; D000495:Allosteric Site; D005635:Fructosediphosphates / Q000494:pharmacology*; D006598:Hexosediphosphates / Q000494:pharmacology*; D007700:Kinetics; D008954:Models, Biological; D009090:Mucorales / Q000201:enzymology*; D010800:Phycomyces / Q000201:enzymology*; D011770:Pyruvate Kinase / Q000378:metabolism*
D005635:Fructosediphosphates; D006598:Hexosediphosphates; D011770:Pyruvate Kinase; C029063:fructose-1,6-diphosphate; D000409:Alanine