Influence of pH on the regulatory kinetics of rat liver phosphofructokinase: a thermodynamic linked-function analysis.
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The relationship between pH and the MgATP inhibition of rat liver phosphofructokinase has been quantiatively evaluated by utilization of a thermodynamic linked-function approach. This approach obviates the need to presuppose discrete inhibited and active states of the enzyme. The behavior of the apparent Michaelis constant for fructose 6-phosphate (Fru-6-P) over a 100-fold concentration range of MgATP conforms to the behavior predicted by the linked-function theory in that, a high concentrations of MgATP, saturation of the inhibitory effect is achieved, a result not predicted by a mutually exclusive two-state model. This behavior is described by the relationship Ka = Ka0[(Kix0 + [X])]/(Kix0 + Q[X])], where Ka is the apparent Michaelis constant for Fru-6-P, Ka0 is the Michaelis constant for Fru-6-P in the absence of MgATP, Kix0 is the dissociation constant of MgATP in the absence of Fru-6-P, and Q is the coupling term that quantitatively describes the finite degree of antagonism between MgATP and Fru-6-P. The free energy of interaction between MgATP and Fru-6-P, obtained from Q, is 1.9 kcal/mol at 25 degrees C. Ka0 and Kix0 are 0.17 and 0.3 mM, respectively. The influence of pH on these three parameters was then systematically investigated, and only Ka0 increased substantially with decreasing pH. Consequently, it is concluded that decreasing the pH does not increase the apparent Ka for Fru-6-P by augmenting the binding or inhibition by MgATP to a significant extent but rather by directly affecting the intrinsic affinity of the enzyme for Fru-6-P. The pK for this effect is 8.1.
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