Thermodynamic linked-function analysis of Mg2+-activated yeast pyruvate kinase

Yeast pyruvate kinase (YPK) is regulated by intermediates of the glycolytic . pathway [eg., phosphoenolpyruvate (PEP), fructose 1,6-bisphosphate (FBP), and citrate] and by the ATP charge of the cell. Recent kinetic and thermod ynamic data with Mn2+-activated YPK show that Mn2+ mediates the allosteric communication between the substrate, PEP, and the allosteric effector, FBP [Mesecar, A., and Nowak, T. (1997) Biochemistry 36, 6792, 6803]. These resu lts indicate that divalent cations modulate multiligand interactions, and h ence cooperativity with YPK. The nature of multiligand interactions on YPK was investigated in the presence of the physiological divalent activator Mg 2+. The binding interactions of PEP, Mg2+, and FBP were monitored by fluore scence spectroscopy. The binding data were subject to thermodynamic linked- function analysis, to; determine the magnitudes of the multiligand interact ions governing the allosteric activation of YPK. The two ligand coupling fr ee energies between PEP and Mg2+, PEP and FBP, and FBP and Mg2+ are 0.88, - 0.38, and -0.75 kcal/mol, respectively. The two-ligand coupling free energi es, between PEP; and Mn2+ and FBP and Mn2+ are more negative than those wit h Mg2+ as the cation. This indicates that the interactions between the diva lent cation and PEP with YPK are different for Mg2+ and Mn2+ and that the i nteraction is not simply electrostatic in nature, as originally hypothesize d. The magnitude of the, heterotropic interaction between the metal and FBP is similar with Mg2+ and Mn2+. The simultaneous binding of Mg2+, PEP, and FBP to YPK is favored by 3.21 kcal/mol. compared tu independent binding. Th is complex is destabilized by 3.30 kcal/mol relative to the analogous YPK-M n2+-PEP-FDP complex. Interpretation of K-d values when cooperative binding occurs must be done with care as these are not simple thermodynamic constan ts. These data demonstrate that the divalent metal, which, activates, phosp horyl transfer in YPK, plays a key role in modulating the various multiliga nd interactions that define the overall allosteric properties of the enzyme .