Abstract
An equilibrium isotope effect on association of [1-18O]oxamate to form a ternary complex with lactate dehydrogenase and NADH of 0.9840 +/- 0.0027 has been measured by equilibrium dialysis and whole molecule isotope ratio mass spectrometry. Semiempirical calculations of vibrational frequencies using various models for solvent were shown to predict an inverse equilibrium isotope effect on association. However, the calculated effect cannot be directly attributed to one specific normal mode or vibrational force constant. Analysis of the carboxylate interaction with the guanidinium ion showed that the ionic interaction increased the torsional force constant for rotation about the carbon-carbon bond of oxamate. The minimum energy geometry for oxamate interacting with methyl guanidinium predicts that the plane of the oxamate carboxylate 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 characterization of the interaction of ligands with protein active sites.
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