METAL-ION-MEDIATED ALLOSTERIC TRIGGERING OF YEAST PYRUVATE-KINASE .1.A MULTIDIMENSIONAL KINETIC LINKED FUNCTION-ANALYSIS

Regulation of the glycolytic pathway is considered to be primarily ach ieved by the carbon metabolites resulting from glucose metabolism [e.g ., fructose 1,6-diphosphate (FDP), phosphoenolpyruvate (PEP), and citr ate] and by the ATP charge of the cell. The divalent cations (e.g., Mg 2+ and Mn2+) have not been considered as having regulatory roles in gl ycolysis, although they are involved in almost every enzyme-catalyzed reaction in the pathway. Using a kinetic linked-function analysis of s teady-state kinetic data for the interactions of PEP, FDP, and Mn2+ wi th yeast pyruvate kinase(YPK), we have found that the divalent metal i s the principal trigger of the allosteric responses observed with this enzyme, The interaction of Mn2+ to YPK enhances the interaction of FD P by -1.6 kcal/mol and the interaction of PEP by -2.8 kcal/mol. The si multaneous interaction of all three of these ligands to YPK is favored by -4.3 kcal/mol over the sum of their independent binding free energ ies. Surprisingly, the binding of the allosteric activator FDP does no t directly influence the binding of the substrate PEP since a coupling free energy near zero was calculated for these two ligands. Thus, com munication between the PEP and FDP sites occurs structurally through t he metal by an allosteric relay mechanism. These conclusions are suppo rted by results of a thermodynamic linked-function analysis of direct binding data for the interactions of PEP, FDP, and Mn2+ with YPK [Mese car, A. D., & Nowak, T. (1997) Biochemistry (following paper in this s eries)l. Our findings raise important questions as to the possible rol es of divalent metals in modulating multiligand interactions with YPK and in the regulation of the glycolytic pathway.