Kinetic Linked-Function Analysis of the Multiligand Interactions on Mg2+-Activated Yeast Pyruvate Kinase

• Published in: Biochemistry (Easton) 2001-10, Vol.40 (43), p.13097-13106
• Main Authors: Bollenbach, Thomas J, Nowak, Thomas
• Format: Article
• Language: English
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi010126o

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.