Kinetic properties of human muscle pyruvate kinase.

The steady-state kinetics of human skeletal muscle pyruvate kinase (MA) and its RNA-complex (MB) has been examined and compared. Kinetic studies revealed significant differences in kinetic properties with respect to free and complex form of pyruvate kinase. The MA form follows a simple Michaelis-Menten kinetics in contrast with the MB form, which displays a negative cooperativity with respect to ADP. Vmax for the complex is 40-60% that for free enzyme. Heterologous RNA is a noncompetitive inhibitor of free enzyme but the kinetics of the complex (MB) is not affected. In presence of 1.0 mM ATP in an assay mixture the kinetic constants of the complex were unchanged except for Vmax, which increased by nearly 60%. Aged preparations of free enzyme (MA) were activated by 100% and more, but the native enzyme was inhibited by 22%. Inorganic phosphate is a potent activator of both forms of pyruvate kinase. In presence of 50 mM K-phosphate the apparent Michaelis constant and interaction coefficient are unchanged, but Vmax for free enzyme increases by 35% and for the complex by 70%, respectively. The specific activity of aged MA form can be restored to the original value after incubation of the enzyme in 50 mM K-phosphate, pH 7.6, or by addition of ATP (1.0 mM) to the assay mixture.