Secondary nitrogen-15 isotope effects on the reactions catalyzed by alcohol and formate dehydrogenases

Abstract

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.

Theoretical Investigation of the Hydride Transfer from Formate to NAD+ and the Implications for the Catalytic Mechanism of Formate Dehydrogenase

“…29 Similarly, the calculated secondary kinetic isotope effect for 15 N1 gives a value of 1.0042, which is comparable to the experimental value of 1.004 ( 0.001 for hydride transfer from formate to an acetyl-substituted NAD + -model fragment bound to the enzyme. 16 Clearly, these results are in good agreement with the experimental data. This calculated normal value of the secondary 15 N1 kinetic isotope effect further strengthens the proposal that the nitrogen center of the pyridine moiety remains planar in the transition state, 16 as it indicates that the hybridization of N1 is unchanged between the reactants and the transition state.…”

“…16 Clearly, these results are in good agreement with the experimental data. This calculated normal value of the secondary 15 N1 kinetic isotope effect further strengthens the proposal that the nitrogen center of the pyridine moiety remains planar in the transition state, 16 as it indicates that the hybridization of N1 is unchanged between the reactants and the transition state. Furthermore, the great similarity between the kinetic isotope effects calculated for the gas-phase reaction and the experimental kinetic isotope effect in the enzyme suggests that the transition state for formate oxidation in the enzyme closely resembles the gas-phase transition state structure.…”

NAD+-dependent formate dehydrogenase

“…It was concluded that hydride transfer taking place in the central ternary complexes is fully rate limiting in the case of these FDHs. A relatively low value of the primary isotope effect observed with NADI (Eo' = -320 mV) as coenzyme was attributed by Cleland and co-workers [62,63,76] to the late transition state of the FDH reaction. The transition state becomes progressively earlier with a change in the redox potential of the coenzyme and is nearly symmetrical in the case of acetylpyridine-NAD+ (Eo'= -258 mV).…”

“…Determination of the secondary deuterium as well as multiple heavy atom (13C, 180, 15N) isotope effects resulted in the formulation of the structure of the transition state ( Figure 2) of the reaction for C. boidinii FDH [62,63,76]. Its main features are: (1) the transition state is linear, and in the case of NADI resembles the products (late transition state); (2) the bending motion of the secondary hydrogen is coupled in the transition state to hydride transfer; and (3) the contribution of tunnelling of both hydrogens in the transition state is significant.…”

“…Based on the substantial observed secondary "5N isotope effect, a boat conformation of the pyridine ring of NADI was originally suggested as the means of raising the carbonium ion character on C-4 and facilitating hydride transfer [63]. However, the values for these gross effects for FDH and ADH were reconsidered [76], and there seems to be no pyramidalization of the N-I atom of the nicotinamide moiety in the coenzyme in the transition state. Thus the pyridine ring of the nucleotide remains planar in the transition state in these reactions.…”