Determination of the kinetic and chemical mechanism of malic enzyme using (2R,3R)-erythro-fluoromalate as a slow alternate substrate

(2R,3R)-erythro-Fluoromalate, but not the three isomer, is a slow substrate for chicken liver malic enzyme with either NADP or 3-acetylpyridine-NADP ( APADP) as the other substrate. The K-m for erythro-fluoromalate is similar to that of malate, but the turnover number with NADP is 3300-fold slower, a lthough 5.5-fold faster with APADP than with NADP. Deuteration of fluoromal ate nt C-2 gave an isotope effect on V/K of 1.39 with NADP and 3.32 with AP ADP. With NADP, the C-13 isotope effects at C-3 were 1.0490 with unlabeled and 1.0364 with deuterated fluoromalate. With APADP, the corresponding valu es were 1.0138 and 1.0087. These data show that the mechanism is stepwise w ith both nucleotide substrates, in contrast to the reaction of malate and A PADP, which was postulated to be concerted by Karsten et al. [Karsten, W. E ., and Cook, P. F. (1994) Biochemistry 33, 2096-2103], a conclusion recentl y shown to be comet by Edens et al. [Edens, W. A., Urbauer, J. L., and Clel and, W. W. (1997) Biochemistry 36, 1141-1147]. To explain the effect of deu teration on the C-13 isotope effect with APADP, it is necessary to assume a secondary C-13 isotope effect at C-4 on the hydride transfer step of simil ar to 1.0064 (assuming 5.7 as the intrinsic primary deuterium isotope effec t and 1.054 as the product of the C-13 equilibrium isotope effect on hydrid e transfer and the intrinsic C-13 isotope effect on decarboxylation). The s econdary C-13 isotope effect on hydride transfer is thought to result from hyperconjugation between the carbonyl group and C-4 of the enzyme-bound flu orooxaloacetate intermediate.