Kinetic linked-function analysis of the multiligand interactions on Mg2+-activated yeast pyruvate kinase

The multiligand interactions governing the allosteric response of Mg2+-activated yeast pyruvate kinase (YPK) during steady-state turnover were quantitated by kinetic linked-function analysis. The substrate, PEP, the enzyme-bound divalent metal, Mg2+, and the allosteric effector, FBP, positively influence each other's interaction with the enzyme in the presence of saturating concentrations of the second substrate, MgADP. The presence of Mg2+ enhances the interaction of PEP and of FBP with YPK by -2.0 and -1.0 kcal/mol, respectively. The simultaneous interaction of PEP, Mg2+, and FBP with YPK is favored by -4.1 kcal/mol over the sum of their independent binding free energies. The coupling free energies measured for Mg2+-activated YPK are weaker than the corresponding coupling free energies measured for Mn2+-activated YPK [Mesecar, A., and Nowak, T. (1997) Biochemistry 36, 6792, 6803], but are consistent with results of thermodynamic measurements with the Mg2+-YPK complex [Bollenbach, T. J., and Nowak, T. (2001) Biochemistry 36,13088-13096]. A comparison of ligand binding data measured by kinetic and thermodynamic linked-function analyses reveals that the MgADP complex modulates both the binding of the other three ligands and the two- and three-ligand coupling interactions between the other three ligands. Enzyme-bound Mg2+ does not influence the homotropic cooperativity in PEP binding to YPK. It is the MgADP complex that induces homotropic cooperativity in PEP binding. It is the enzyme-bound Mn2+ that induces homotropic binding of PEP with Mn2+-activated YPK. These results lend support to the hypothesis that divalent metals modulate the interactions of ligands on YPK and that divalent metals play a role in regulation of the glycolytic pathway.