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Studies on Heart Phosphofructokinase

BINDING PROPERTIES OF NATIVE ENZYME AND OF ENZYME DESENSITIZED TO ALLOSTERIC CONTROL

From the ¹ From the Department of Pharmacology, Stanford University School of Medicine, Stanford, California 94305

The binding properties of purified sheep heart phosphofructokinase were studied with a gel filtration technique and were compared with those of enzyme desensitized to allosteric control by photooxidation. The enzyme, after treatment with charcoal, had a tendency to aggregate and to be retained on Sephadex columns. Binding studies, therefore, had to be carried out in the presence of different substrates, sulfate or phosphate salts, to obtain maximum enzyme recovery. The native enzyme binds, at saturation, 3.6 moles of ATP and 2.0 moles of citrate per 100,000 g. ATP inhibits binding of the enzyme to citrate. The binding of fructose-6-P is dependent on protein concentration. The enzyme binds as much as 11.8 moles of fructose-6-P per 100,000 g. Much of this binding is on sites which are nonspecific. In the presence of inorganic phosphate the nonspecific binding was reduced to a minimum, and the enzyme binds only 1.75 moles of fructose-6-P per 100,000 g. The photooxidized enzyme binds at saturation 2 moles of ATP, 2 moles of fructose-6-P per 100,000 g, and no citrate. The results indicate that there are two catalytic sites and two allosteric sites per 100,000 g. ATP, therefore, besides being a substrate, is also a modifier which inhibits the enzyme by binding at a site different from the catalytic site, namely the allosteric site. Citrate, like ATP, inhibits phosphofructokinase by binding at the same allosteric site.

The effect of different enzyme effectors was tested on the binding of the above mentioned substrates. Fructose-1,6-di-P decreases the affinity of both native and photooxidized enzyme for ATP binding. The hexose diphosphate does not affect the affinity of the native enzyme to citrate binding but reduces the number of its binding sites for citrate. ß-Glycerophosphate, as well as inorganic phosphate, reduces the binding of ATP to the enzyme. The adenylic nucleotides, inorganic phosphate, and ß-glycero-P inhibit citrate binding. Cyclic 3',5'-AMP is the most potent of these agents. The results are discussed in connection with the allosteric kinetics and other physicochemical properties of phosphofructokinase.

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