Interaction of calmodulin with muscle phosphofructokinase. Interplay with metabolic effectors of the enzyme under physiological conditions.
The hysteretic calmodulin-induced inactivation of muscle phosphofructokinase and the calmodulin-mediated reactivation are essentially dependent on environmental conditions. The interplay of calmodulin during these reactions and at allosteric conditions with Mg . ATP, fructose 6-phosphate, adenosine 5'-[beta, gamma-imido]triphosphate and with the allosteric effectors AMP, ADP, fructose 1,6-bisphosphate, fructose 2,6-bisphosphate and glucose 1,6-bisphosphate was studied by two techniques. (a) A two-step technique with a preincubation of enzyme, calmodulin and effectors in close to physiological concentrations before dilution into an optimal activity assay. It reveals aggregation and slowly reversible conformation changes. (b) A direct assay of dilute enzyme at allosteric conditions. Dominating in the interplay of calmodulin with metabolic effectors is the competitive-like action of calmodulin on Mg . ATP binding to the regulatory sites of the enzyme. At high enzyme concentrations in the absence of hexose phosphates, i.e. at noncatalytic conditions calmodulin counteracts the stabilization of the highly active tetrameric form caused by Mg . ATP. In the allosteric assay it counteracts the ATP-induced allosteric inhibition. In both cases calmodulin acts synergistic with AMP and ADP. To a minor degree calmodulin also counteracts the stabilization of the tetrameric form caused by fructose 6-phosphate and hexose bisphosphate, now however antagonistically to AMP and ADP. By the demonstrated interactions the enzyme can be slowly and hysteretically shifted between an active tetrameric and an inactive dimeric state under control metabolic conditions and of Ca2+ and calmodulin. Resting conditions will inactivate and high contractile activity reactivate available enzyme.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000494:Allosteric Regulation; D000818:Animals; D002147:Calmodulin / Q000378:metabolism* / Q000494:pharmacology; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D004793:Enzyme Reactivators; D005636:Fructosephosphates / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D010732:Phosphofructokinase-1 / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D011817:Rabbits; D013379:Substrate Specificity
D002147:Calmodulin; D004793:Enzyme Reactivators; D005636:Fructosephosphates; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1