EQUILIBRIUM ISOTOPE EFFECT ON TERNARY COMPLEX-FORMATION OF [1-O-18]OXAMATE WITH NADH AND LACTATE-DEHYDROGENASE

An equilibrium isotope effect on association of [1-O-18]oxamate to for m a ternary complex with lactate dehydrogenase and NADH of 0.9840 +/- 0.0027 has been measured by equilibrium dialysis and whole molecule is otope ratio mass spectrometry. Semiempirical calculations of vibration al frequencies using various models for solvent were shown to predict an inverse equilibrium isotope effect on association. However, the cal culated effect cannot be directly attributed to one specific normal mo de or vibrational force constant. Analysis of the carboxylate interact ion with the guanidinium ion showed that the ionic interaction increas ed the torsional force constant for rotation about the carbon-carbon b ond of oxamate. The minimum energy geometry for oxamate interacting wi th methyl guanidinium predicts that the plane of the oxamate carboxyla te will be at an oblique angle to the plane defined by the guanidinium nitrogens. The combination of experimental and calculated equilibrium isotope effects on association holds the potential to improve the cha racterization of the interaction of ligands with protein active sites.