Relationship between fructose 2,6-bisphosphate activation and MgATP inhibition of rat liver phosphofructokinase at high pH. Kinetic evidence for individual binding sites linked by finite couplings.
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The concentration of fructose 6-phosphate required to produce half-maximal velocity of rat liver phosphofructokinase at pH 9 (Ka) has been measured at 110 different combinations of MgATP and fructose 2,6-bisphosphate (Fru-2,6-BP) concentrations spanning the range 0.1-100 mM and 0.003-100 microM, respectively. The data have been evaluated by nonlinear regression to an equation resulting from a linked-function analysis of an enzyme capable of binding three ligands simultaneously at separate sites. In addition, the data have been fit to equations, derived from the linked-function expression, that would result if various combinations of antagonistic ligands were unable to bind to the enzyme simultaneously, even at high concentration, either because they compete for a single binding site or because they bind exclusively to different conformational forms of the enzyme. The complete linked-function equation is able to predict the Ka for rat liver phosphofructokinase as a function of any Fru-2,6-BP and/or MgATP concentration significantly better than any of the alternatives examined, particularly at high concentrations of one or both modifier ligands. The free energy couplings between all three possible pairs of ligands are of quite moderate magnitude, especially when the multiplicity of binding sites for each ligand that actually exists on the functional enzyme is considered. Therefore, we conclude that any explanation of the action of Fru-2,6-BP and MgATP by a model more elaborate than the simple linked-function case considered herein cannot be simplified by assuming that the properties of rat liver phosphofructokinase result from an equilibrium of limiting conformational states that exhibit exclusive binding properties.
author list (cited authors)
Reinhart, G. D., & Hartleip, S. B
complete list of authors
Reinhart, GD||Hartleip, SB