Metal-Ion-Mediated Allosteric Triggering of Yeast Pyruvate Kinase 2. A Multidimensional Thermodynamic Linked-Function Analysis

Abstract

A role has been proposed for the free divalent metal in triggering the allosteric responses of yeast pyruvate kinase based upon a kinetic linked-function analysis [Mesecar, A. D., & Nowak, T. (1997a) (preceding paper in this series)]. The major conclusion from the analysis is that the allosteric activator, fructose 1,6-diphosphate (FDP), does not directly communicate with the substrate, phosphoenolpyruvate (PEP), at the active site of the enzyme: it is Mn2+ that mediates the allosteric communication between the PEP and FDP sites in an allosteric relay mechanism. Assumptions were necessary to treat kinetic parameters as thermodynamic parameters, and the presence of the substrate ADP was necessary for the kinetic analysis. In this study, the influence of FDP on the interactions of PEP and Mn2+ and the influence of PEP and Mn2+ on the interaction of FDP with YPK were measured, where possible, by direct binding methods in the absence of ADP. Direct binding data were then subjected to a thermodynamic linked-function analysis for a heterotropic, three ligand coupled system in order to ascertain the two and three ligand coupling free energies. The two ligand coupling free energies deltaG(Mn-PEP), deltaG(Mn-FDP), and deltaG(PEP-FDP) are -3.88, -1.09, and -0.22 kcal/mol, respectively. These values indicate that positive, heterotropic interactions exist between each of these ligand pairs. The three ligand coupling free energy term, deltaG(Mn-PEP-FDP), indicates that simultaneous binding of Mn2+, PEP, and FDP is considerably favored over the sum of their independent binding free energies by -6.6 kcal/mol. These results demonstrate the key role of the metal in the modulation of ligand binding and are consistent with the values and the relationships of the kinetic parameters obtained from the kinetic linked-function analysis.

The Proton Transfer Step Catalyzed by Yeast Pyruvate Kinase

“…The single tryptophan, Trp-452, was selectively excited at 295 nm so that observed changes in the intrinsic fluorescence could be correlated with conformational changes at a specific localized region in YPK. The apo forms of T298S and T298A YPK have a tryptophan emission maximum at ϳ334 nm, similar to that for apo wild type YPK (16).…”

“…A slight red shift in the fluorescence emission spectrum was observed for the T298A⅐Mn 2ϩ complex. In wild type YPK, Mn 2ϩ binding causes a 2 nm blue shift and a fluorescence quenching of 9% (16). Saturation of the YPK⅐Mn 2ϩ complex with PEP caused 32% fluorescence quenching and a 2 nm blue shift in T298S and 36% quenching in T298A.…”

“…Wild type, T298S, and T298A yeast pyruvate kinases were purified as described by Mesecar and Nowak (16). PEP, ADP, Fru-6-P 2 , disodium NADH, glycerol, Dowex 1 chloride (400-mesh), and buffers were purchased from Sigma.…”

“…Transformed pyk1-5 containing pPYK101 with the T298S and T298A mutations were grown on glucose minimal media containing the following per liter: 6.7 g of yeast nitrogen base, 10 of g succinic acid, 4 of g NaOH, 2% glucose, 0.5% casamino acids, 0.001% adenine, and 0.001% methionine. Cells were harvested, and the wild type YPK and T298S and T298A mutants were purified by the procedure published previously (16). Yeast pyk1-5 containing the T298V PYK101 was grown on glycerol-ethanol minimal media prepared the same as for T298S and T298A except containing 2% glycerol and 2% ethanol in place of 2% glucose.…”

The Negative Dominant Effects of T340M Mutation on Mammalian Pyruvate Kinase

“…The apparent cooperativity, quantitated by the Hill coefficient (n), was not significantly affected by the mutation. Recent reports showed that the interactions of PEP and Mg 2ϩ with PK is coupled (19,20). In view of the significant change in the value of K app,PEP , the effect of Mg 2ϩ concentration was studied by monitoring PK activity at fixed concentrations of 2 mM ADP and 20 mM PEP and varying concentrations of MgSO 4 .…”