Steady-state kinetic studies of muscle pyruvate kinase were conducted as a function of pH and phenylalanine concentrations. Results show that at a pH below 7.0, there is no observable effect of phenylalanine on the kinetic properties of muscle pyruvate kinase. When the results at a pH below 6.5 are used as the state for comparison, the kinetic results show that phenylalanine and proton exert a synergistic effect on the allosteric properties of the enzyme. A significantly greater change in Hill coefficients at high pH can be detected in the presence of phenylalanine than in its absence. To pinpoint the specific mechanism that leads to the synergistic effect, the kinetic data were resolved into the five equilibrium and two rate constants that characterize the basic two-state model. It can be shown that [formula omitted] the binding constant of phenylalanine to the inactive T state, is strongly proton-linked. The affinity of phenylalanine for the T state increases with increasing pH. When the pH dependence of [formula omitted] was analyzed by the linked-function theory [Wyman, J. (1964) Adv. Protein Chem. 19, 224-285], it was shown that deprotonation favors phenylalanine binding to the T state. [formula omitted] (the binding constant of phenylalanine to the active R state), [formula omitted] (the binding constant of substrate to the T state), and L (the isomerization constant of the two states) not only are all weakly proton-linked but also it was shown that protonation favors the ligand–pyruvate kinase complex. [formula omitted], the binding constant of substrate for the R state, shows no observable linkage to proton concentration. Thus, pH exhibits differential effects on these equilibrium constants both qualitatively and quantitatively. Knowing the proton linkage relationships, it is possible to conclude that the synergistic effect of phenylalanine and proton can be explained by the interplay among the strong proton-linked effect on the affinity of phenylalanine to the T state and the apparently weak or insignificant proton linkage in the other equilibrium parameters.
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