Determination of the chemical mechanism of malic enzyme by isotope effects

Carbon-13 isotope effects have been determined for all four carbons of L-malate as a substrate for chicken liver malic enzyme, using either NADP or acetylpyridine-NADP as the other substrate. The effect of deuteration at C2 of malate was then used to tell whether the chemical mechanism of this oxidative decarboxylation was stepwise, with oxaloacetate as an intermediate, or concerted. With NADP, the C-13 isotope effects at C3 and C4 both decrease with deuteration of malate, showing a stepwise mechanism, as previously determined [Hermes, J. D., Roeske, C. A., O'Leary, M. H., & Cleland, W. W, (1982) Biochemistry 21, 5106-5114]. With acetylpyridine-NADP, however, the C-13 isotope effects at both C3 and C4 increase with deuteration of malate. While the increase at C4 could be explained by a secondary C-13 isotope effect on hydride transfer, the increase at C3 proves that the chemical mechanism has changed to a concerted one, presumably because hydride transfer is more rate-limiting and the overall equilibrium constant is more favorable by 2 orders of magnitude. The transition state for this concerted reaction is asynchronous, however, with an intrinsic deuterium isotope effect of similar to 5 and a C-13 isotope effect of only 1.010-1.015. Equilibrium C-13 isotope effects for conversion of carbons 2, 3, and 4 of malate to pyruvate or CO2 are 1.010, 1.011, and 0.988, respectively. Measured C-13 isotope effects at C2 of malate are slightly inverse, but no explanation for this is obvious. With NADP, deuterium isotope effects at C3 of 1.17 and 1.08 for di- and monodeuteration and an increase in the C-13 isotope effect at C4 upon dideuteration at C3 are consistent with a stepwise mechanism with the deuterium isotope effect at C3 being only on the decarboxylation step. Smaller deuterium isotope effects of 1.03-1.04 from dideuteration at C3 with acetylpyridine-NADP are consistent with a concerted but asynchronous mechanism where C-C cleavage is not far advanced in the transition state.