ALLOSTERIC PROPERTIES OF MUSCLE PHOSPHOFRUCTOKINASE .I. BINDING OF MAGNESIUM ADENOSINE TRIPHOSPHATE TO INHIBITORY SITE

Magnesium ions plus adenosine 5′-triphosphate protected the most reactive thiol group of rabbit skeletal muscle phosphofructokinase from reaction with dithiobis(2-nitrobenzoic acid). The reactivity of this thiol group appeared to be an indicator for the binding of magnesium adenosine 5′- triphosphate to the inhibitory site on the enzyme. Employing thiol reactivity as a means of measuring the relative saturation of the enzyme, the following properties of the magnesium adenosine 5′-triphosphate binding site were observed. (1) The dissociation constant for magnesium adenosine 5′-triphosphate was lower than 10 μM. The affinity of the enzyme for adenosine triphosphate alone at the site being monitored was more than 100 times less than that for the metal complex and the affinity for magnesium inosine 5′-triphosphate was approximately 25 times less than that for magnesium adenosine 5′-triphosphate. (2) Adenosine 5′-monophosphate, adenosine 3′,5′-cyclic monophosphate, and inorganic phosphate reduced the affinity of the enzyme for magnesium adenosine 5′-triphosphate. (3) Citrate increased the affinity of the enzyme for the metal complex. (4) The dissociation constant for magnesium adenosine 5′-triphosphate was much higher at pH 7.35 than at pH 7.0. (5) Magnesium β,γ-methyleneadenosine 5′-triphosphate was also capable of binding to the inhibitory site. In the presence of fructose 6-phosphate the binding of derivative was decreased. The results of these studies are discussed in relation to the kinetics of phosphofructokinase and in relation to present views of allosteric properties of enzymes. © 1969, American Chemical Society. All rights reserved.