Thermodynamic Linked-Function Analysis of Mg²⁺-Activated Yeast Pyruvate Kinase

Abstract

Yeast pyruvate kinase (YPK) is regulated by intermediates of the glycolytic pathway [e.g., phosphoenolpyruvate (PEP), fructose 1,6-bisphosphate (FBP), and citrate] and by the ATP charge of the cell. Recent kinetic and thermodynamic data with Mn(2+)-activated YPK show that Mn(2+) mediates the allosteric communication between the substrate, PEP, and the allosteric effector, FBP [Mesecar, A., and Nowak, T. (1997) Biochemistry 36, 6792, 6803]. These results indicate that divalent cations modulate multiligand interactions, and hence 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, Mg(2+), and FBP were monitored by fluorescence spectroscopy. The binding data were subject to thermodynamic linked-function analysis to determine the magnitudes of the multiligand interactions governing the allosteric activation of YPK. The two ligand coupling free energies between PEP and Mg(2+), PEP and FBP, and FBP and Mg(2+) are 0.88, -0.38, and -0.75 kcal/mol, respectively. The two-ligand coupling free energies between PEP and Mn(2+) and FBP and Mn(2+) are more negative than those with Mg(2+) as the cation. This indicates that the interactions between the divalent cation and PEP with YPK are different for Mg(2+) and Mn(2+) and that the interaction is not simply electrostatic in nature, as originally hypothesized. The magnitude of the heterotropic interaction between the metal and FBP is similar with Mg(2+) and Mn(2+). The simultaneous binding of Mg(2+), PEP, and FBP to YPK is favored by 3.21 kcal/mol compared to independent binding. This complex is destabilized by 3.30 kcal/mol relative to the analogous YPK-Mn(2+)-PEP-FDP complex. Interpretation of K(d) values when cooperative binding occurs must be done with care as these are not simple thermodynamic constants. These data demonstrate that the divalent metal, which activates phosphoryl transfer in YPK, plays a key role in modulating the various multiligand interactions that define the overall allosteric properties of the enzyme.

The Proton Transfer Step Catalyzed by Yeast Pyruvate Kinase

“…Binding of the allosteric activator Fru-6-P 2 to all the enzyme complexes studied has been altered upon mutation of the active site Thr-298. The effects are metal-dependent, reinforcing the fact that the coupling between the Fru-6-P 2 and PEP sites in yeast PK is modulated through the enzyme-bound metal (16,28). Again, it is evident that small conformational changes introduced at the active site of YPK by mutation of Thr-298 to serine or alanine can induce long range effects at the Fru-6-P 2 -binding site situated more than 40 Å away.…”

“…The conformational location of the two residues Thr-296 and Arg-310 (Thr-298 and Arg-312 in YPK numbering) appears to play an important role in the T-state to R-state allosteric transition. Previous studies (16,28) have shown that binding of Mn 2ϩ or Mg 2ϩ causes a conformational change that favors the communication between the PEP and Fru-6-P 2 sites. A plausible theory that emanates from the study of Rigden et al (27) is that the binding of PEP to PK elicits a change in conformation of Thr-298 (YPK numbering).…”

“…Two separate signals may be necessary to determine an allosteric transition from the catalytic site of PK, one from the enzyme-bound divalent activator and the second from PEP via Thr-298. The interaction between the M 2ϩ activator and PEP with YPK is highly coupled (16,28). The role of Thr-298 in the reaction catalyzed by YPK seems to be complex and involves both catalytic and regulatory functions.…”

“…The intrinsic fluorescence of Trp-452 is quenched upon ligand binding, allowing for the measurement of thermodynamic dissociation constants of ligands from various YPK complexes (16,28 (30). In the latter case, it was proposed that the cooperative binding of Br-PEP to wild type YPK is masked by a late slow kinetic step and therefore cooperativity in kinetics is not observed.…”

Regulation of Yeast Pyruvate Kinase by Ultrasensitive Allostery Independent of Phosphorylation

“…However, prior studies indicated that FBP activates Cdc19 but not in a cooperative manner, i.e., with a Hill coefficient approximately equal to 1. These prior in vitro studies, however, measured enzymatic activity with FBP and PEP at non-physiological concentrations (Mesecar and Nowak, 1997a, b; Nowak and Bollenbach, 2001; Susan-Resiga and Nowak, 2004). While the cellular concentrations of PEP range from 0.03 to 0.8 mM, PEP was typically added in saturating concentrations of 20 mM to facilitate the enzyme activity measurements.…”