Rat liver pyruvate kinase: Influence of ligands on activity and fructose 1,6-bisphosphate binding

Abstract

The ability for various ligands to modulate the binding of fructose 1,6-bisphosphate (Fru-1,6-P 2) with purified rat liver pyruvate kinase was examined. Binding of Fru-1,6-P 2 with pyruvate kinase exhibits positive cooperativity, with maximum binding of 4 mol Fru-1,6-P 2 per enzyme tetramer. The Hill coefficient n H), and the concentration of Fru-1,6-P 2 giving half-maximal binding [ FBP] 1 2 , are influenced by several factors. In 150 m m Tris-HCl, 70 m m KCl, 11 m m MgSO 4 at pH 7.4, [ FBP] 1 2 is 2.6 μ m and n H is 2.7. Phosphoenolpyruvate and pyruvate enhance the binding of Fru-1,6-P 2 by decreasing [ FBP] 1 2 . ADP and ATP alone had little influence on Fru-1,6-P 2 binding. However, the nucleotides antagonize the response elicited by pyruvate or phosphoenolpyruvate, suggesting that the competent enzyme substrate complex does not favor Fru-1,6-P 2 binding. Phosphorylation of pyruvate kinase or the inclusion of alanine in the medium, two actions which inhibit the enzyme activity, result in diminished binding of low concentrations of Fru-1,6-P 2 with the enzyme. These effectors do not alter the maximum binding capacity of the enzyme but rather they raise the concentrations of Fru-1,6-P 2 needed for maximum binding. Phosphorylation also decreased the n H for Fru-1,6-P 2 binding from 2.7 to 1.7. Pyruvate kinase activity is dependent on a divalent metal ion. Substituting Mn 2+ for Mg 2+ results in a 60% decrease in the maximum catalytic activity for the enzyme and decreases the concentration of phosphoenolpyruvate needed for half-maximal activity from 1 to 0.1 m m. As a consequence, Mn 2+ stimulates activity at subsaturating concentrations of phosphoenolpyruvate, but inhibitis at saturating concentrations of the substrate or in the presence of Fru-1,6-P 2. Both Mg 2+ and Mn 2+ diminish binding of low concentrations of Fru-1,6-P 2; however, the concentrations of the metal ions needed to influence Fru-1,6-P 2 binding exceed those needed to support catalytic activity.