DETERMINATION OF THE CHEMICAL MECHANISM OF MALIC ENZYME BY ISOTOPE EFFECTS

Citation
Wa. Edens et al., DETERMINATION OF THE CHEMICAL MECHANISM OF MALIC ENZYME BY ISOTOPE EFFECTS, Biochemistry, 36(5), 1997, pp. 1141-1147
Citations number
14
Categorie Soggetti
Biology
Journal title
ISSN journal
00062960
Volume
36
Issue
5
Year of publication
1997
Pages
1141 - 1147
Database
ISI
SICI code
0006-2960(1997)36:5<1141:DOTCMO>2.0.ZU;2-C
Abstract
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.