A hypothetical model of the influence of inorganic phosphate on the kinetics of pyruvate kinase.

This paper presents a simple solution to the problem of approximating the calculated curve of reaction progress to the measured curve which is usually disturbed by initial oscillation of auxiliary lactate dehydrogenase (LDH) reaction. The experiments leading to the determination of the apparent Km for phosphoenolpyruvate (PEP) and Vm were performed. For precise estimation of kinetic parameters (Km and Vm) of the M1 isozyme of pyruvate kinase (PK), measured by coupling it to LDH reaction, the sequence of Michaelis-Menten for pyruvate kinase and second-order kinetics for lactate dehydrogenase reaction as well as a non-zero initial concentration of lactate was assumed. The functions of apparent Km and Vm of pyruvate kinase with respect to phosphate concentration, computed by an analysis of the total reaction progress curves, indicate that the reaction mixture contains an uncompetitive inhibitor of pyruvate kinase, and that the phosphate binds this inhibitor. The proposed simple mathematical model of pyruvate kinase Km and Vm increase by inorganic phosphate assumes that the pyridine nucleotides (NAD-derivatives) are kinase inhibitors. An approximate dissociation constant for pyridine nucleotides-phosphate complex and true Km of pyruvate kinase for PEP were estimated. The proposed model fits exactly the entire measured reaction process.