The variation of the C-D vibrational stretching frequency in primary and secondary alcohols containing the D-C-O-H functionality has been examined for cases in which the alcohol functions as a proton donor in an H-bond. The C-D stretching frequency is a function of the H-bond enthalpy of formation determined by Hartree-Fock calculations, decreasing by approximately 5 cm -1 per kcal/mol. This decrease in frequency is attributed to the increase in the overlap of the O-H bonding electrons with the C-D antibonding orbital as the H-bond is strengthened. The Raman spectra of [1-D]trifluoroethanol and [1-D]trifluoroethoxide in aqueous solution serve as an example; the alcohol has two separate C-D stretches that differ by 45 cm -1 and deprotonation results in an average 78 cm -1 decrease in the C-D stretching frequency. A measured deuterium equilibrium isotope effect on the acid ionization constant of [1-D 2 ]trifluoroethanol of 1.13 is consistent with a decreased fractionation factor of the C-1 protons due to the decrease in the C-D stretching frequency.A model nucleoside complexed with the H-bonding residues at the active site of nucleoside hydrolase indicates that H-bond formation can explain the anomalous secondary isotope effects reported for the hydrolysis of [5′-3 H]inosine (Horenstein et al. Biochemistry 1991, 30, 10788-10795). The correlations of both the C-D stretching frequency and the fractionation factor with the conformation and H-bond strength with primary and secondary alcohols as donors should serve as tools for the characterization of these important interactions in biological systems.

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.

Kinetic isotope effects on association have been measured using the remote label methodology developed by O'Leary and Marlier (1979). The isotope effect on V/KA for the first substrate in an obligatorily ordered mechanism is an isotope effect on its second-order rate constant for association with the enzyme. With phosphoenolpyruvate carboxylase the 18(V/KPEP) when the bridging O is labeled decreases from 1.0056 +/- 0.0007 to 0.9943 +/- 0.0002 as the concentration of bicarbonate, the second substrate, increases from 2 to 200 mM. With pyruvate kinase the 18(V/KPEP) decreases from 1.0024 +/- 0.0014 to 0.9928 +/- 0.0027 as the concentration of ADP increases from 1.5 to 30 mM. These inverse kinetic isotope effects are best understood as arising from an isotope effect on the rate constant for forming the Michaelis complex of enzyme and substrate. The inverse value suggests that the bridging oxygen is in a vibrationally stiffer environment in the transition state for the association reaction.