Calorimetric studies of the role of magnesium ions in yeast enolase catalysis.
The binding of magnesium ions and of the competitive inhibitor 3-phospho-D-glyceric acid to yeast enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11) has been studied calorimetrically. Thermal titration of the apoprotein with magnesium ions provides evidence that two magnesium ions bind immeasurably tightly to the dimeric enzyme, either anticooperatively to interacting sites or to two independent, nonidentical sites. Measurements of the saturation heat in buffers with different enthalpies of protonation are consistent with the release of two protons when the metal-binding sites are filled at pH 7.5. The enthalpy of binding of the two magnesium ions, corrected for the release of two protons, is +11.7 kcal (+49.0 kJ) per mole of dimeric protein. Thermal titration of the magnesium-saturated enzyme with 3-phosphoglyceric acid corroborates the conclusion of Spring and Wold [Biochemistry (1971) 10, 4655-4660] that the enolase dimer possesses two equivalent and independent substrate-binding sites. The dissociation constant for the enzyme-inhibitor complex calculated from the thermal data is 2 mM. The thermal studies of 3-phosphoglyceric acid binding also confirm that metal ions are required for substrate binding and that substrate binds at the two specific metal-binding sites on the apoprotein. Experiments in buffers with different enthalpies of ionization provide evidence for proton uptake when 3-phosphoglyceric acid is bound.
D001059:Apoproteins / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D002151:Calorimetry; D002384:Catalysis; D005994:Glycerophosphates / Q000378:metabolism / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism*; D010751:Phosphopyruvate Hydratase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D001059:Apoproteins; D005994:Glycerophosphates; D010751:Phosphopyruvate Hydratase; D008274:Magnesium