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 deut
eration at C2 of malate was then used to tell whether tile chemical me
chanism of this oxidative decarboxylation was stepwise, with oxaloacet
ate as an intermediate, or concerted. With NADP, the C-13 isotope effe
cts at C3 and C4 both decrease with deuteration of malate, showing a s
tepwise mechanism, as previously determined [Hermes, J. D., Roeske, C.
A., O'Leary, M. H., & Cleland, W. W, (1982) Biochemistry 21, 5106-511
4]. With acetylpyridine-NADP, however, the C-13 isotope effects at bot
h 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 t
ransfer, the increase at C3 proves that the chemical mechanism has cha
nged to a concerted one, presumably because hydride transfer is more r
ate-limiting and the overall equilibrium constant is more favorable by
2 orders of magnitude. The transition state for this concerted reacti
on is asynchronous, however, with an intrinsic deuterium isotope effec
t of similar to 5 and a C-13 isotope effect of only 1.010-1.015. Equil
ibrium C-13 isotope effects for conversion of carbons 2, 3, and 4 of m
alate to pyruvate or CO2 are 1.010, 1.011, and 0.988, respectively. Me
asured C-13 isotope effects at C2 of malate are slightly inverse, but
no explanation for this is obvious. With NADP, deuterium isotope effec
ts 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 consiste
nt with a stepwise mechanism with the deuterium isotope effect at C3 b
eing only on the decarboxylation step. Smaller deuterium isotope effec
ts of 1.03-1.04 from dideuteration at C3 with acetylpyridine-NADP are
consistent with a concerted but asynchronous mechanism where C-C cleav
age is not far advanced in the transition state.