Kinetic linked-function analysis of the multiligand interactions on Mg(2+)-activated yeast pyruvate kinase.
The multiligand interactions governing the allosteric response of Mg(2+)-activated yeast pyruvate kinase (YPK) during steady-state turnover were quantitated by kinetic linked-function analysis. The substrate, PEP, the enzyme-bound divalent metal, Mg(2+), 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 Mg(2+) enhances the interaction of PEP and of FBP with YPK by -2.0 and -1.0 kcal/mol, respectively. The simultaneous interaction of PEP, Mg(2+), 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 Mg(2+)-activated YPK are weaker than the corresponding coupling free energies measured for Mn(2+)-activated YPK [Mesecar, A., and Nowak, T. (1997) Biochemistry 36, 6792, 6803], but are consistent with results of thermodynamic measurements with the Mg(2+)-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 Mg(2+) 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 Mn(2+) that induces homotropic binding of PEP with Mn(2+)-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.
Bollenbach
T J
TJ
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA.
Nowak
T
T
D000244:Adenosine Diphosphate / Q000378:metabolism; D001665:Binding Sites; D004305:Dose-Response Relationship, Drug; D006019:Glycolysis; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008024:Ligands; D008274:Magnesium / Q000378:metabolism*; D008345:Manganese; D010728:Phosphoenolpyruvate / Q000737:chemistry; D011485:Protein Binding; D011770:Pyruvate Kinase / Q000737:chemistry* / Q000378:metabolism*; D013816:Thermodynamics
D008024:Ligands; D008345:Manganese; D000244:Adenosine Diphosphate; D010728:Phosphoenolpyruvate; D007770:L-Lactate Dehydrogenase; D011770:Pyruvate Kinase; D008274:Magnesium