SECONDARY N-15 ISOTOPE EFFECTS ON THE REACTIONS CATALYZED BY ALCOHOL AND FORMATE DEHYDROGENASES

Secondary N-15 isotope effects at the N-1 position of 3-acetylpyridine adenine dinucleotide have been determined, by using the internal competition technique, for horse liver alcohol dehydrogenase (LADH) with cyclohexanol as a substrate and yeast formate dehydrogenase (FDH) with formate as a substrate. On the basis of less precise previous measurements of these N-15 isotope effects, the nicotinamide ring of NAD has been suggested to adopt a boat conformation with carbonium ion character at C-4 during hydride transfer [Cook, P. F., Oppenheimer, N. J., & Cleland, W. W. (1981) Biochemistry 20, 1817]. If this mechanism were valid, as N-1 becomes pyramidal an N-15 isotope effect of up to 2-3% would be observed. In the present study the equilibrium N-15 isotope effect for the reaction catalyzed by LADH was measured as 1.0042 +/- 0.0007. The kinetic N-15 isotope effect for LADH catalysis was 0.9989 +/- 0.0006 for cyclohexanol oxidation and 0.997 +/- 0.002 for cyclohexanone reduction. The kinetic N-15 isotope effect for FDH catalysis was 1.004 +/- 0.001. These values suggest that a significant N-15 kinetic isotope effect is not associated with hydride transfer for LADH and FDH. Thus, in contrast with the deformation mechanism previously postulated, the pyridine ring of the nucleotide apparently remains planar during these dehydrogenase reactions.