Secondary 15N isotope effects on the reactions catalyzed by alcohol and formate dehydrogenases.
Secondary 15N 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 15N 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 15N isotope effect of up to 2-3% would be observed. In the present study the equilibrium 15N isotope effect for the reaction catalyzed by LADH was measured as 1.0042 +/- 0.0007. The kinetic 15N isotope effect for LADH catalysis was 0.9989 +/- 0.0006 for cyclohexanol oxidation and 0.997 +/- 0.002 for cyclohexanone reduction. The kinetic 15N isotope effect for FDH catalysis was 1.004 +/- 0.001. These values suggest that a significant 15N 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.
Rotberg
N S
NS
Institute for Enzyme Research, University of Wisconsin-Madison 53705.
Cleland
W W
WW
D000426:Alcohol Dehydrogenase / Q000378:metabolism*; D000818:Animals; D003511:Cyclohexanols; D005560:Formate Dehydrogenases / Q000378:metabolism*; D006736:Horses; D007553:Isotope Labeling; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009587:Nitrogen Isotopes; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013379:Substrate Specificity
D003511:Cyclohexanols; D009587:Nitrogen Isotopes; D000426:Alcohol Dehydrogenase; D005560:Formate Dehydrogenases