MULTIPLE ISOTOPE EFFECTS WITH ALTERNATIVE DINUCLEOTIDE SUBSTRATES AS A PROBE OF THE MALIC ENZYME REACTION

Deuterium isotope effects and C-13 isotope effects with deuterium- and protium-labeled malate have been obtained for both NAD- and NADP-malic enzymes by using a variety of alternative dinucleotide substrates. With nicotinamide-containing dinucleotides as the oxidizing substrate, the C-13 effect decreases when deuterated malate is the substrate compared to the value obtained with protium-labeled malate. These data are consistent with a stepwise chemical mechanism in which hydride transfer precedes decarboxylation of the oxalacetate intermediate as previously proposed [Hermes, J. D., Roeske, C. A., O'Leary, M. H., & Cleland, W. W. (1982) Biochemistry 21, 51061. When dinucleotide substrates such as thio-NAD, 3-acetylpyridine adenine dinucleotide, and 3-pyridinealdehyde adenine dinucleotide that contain modified nicotinamide rings are used, the C-13 effect increases when deuterated malate is the substrate compared to the value obtained with protium-labeled malate. These data, at face value, are consistent with a change in mechanism from stepwise to concerted for the oxidative decarboxylation portion of the mechanism. However, the increase in the deuterium isotope effect from 1.5 to 3 with a concomitant decrease in the C-13 isotope effect from 1.034 to 1.003 as the dinucleotide substrate is changed suggests that the reaction may still be stepwise with the non-nicotinamide dinucleotides. A more likely explanation is that a beta-secondary C-13 isotope effect accompanies hydride transfer as a result of hyperconjugation of the beta-carboxyl of malate as the transition state for the hydride transfer step is approached. The equality of observed deuterium isotope effects on the V/K values for NADP or NAD and malate shows that addition of these substrates is random rather than ordered as has been previously believed for the NADP enzyme.