(2R,3R)-erythro-Fluoromalate, but not the threo isomer, is a slow substrate for chicken liver malic enzyme with either NADP or 3-acetylpyridine-NADP (APADP) as the other substrate. The Km for erythro-fluoromalate is similar to that of malate, but the turnover number with NADP is 3300-fold slower, although 5.5-fold faster with APADP than with NADP. Deuteration of fluoromalate at C-2 gave an isotope effect on V/K of 1.39 with NADP and 3.32 with APADP. With NADP, the 13C isotope effects at C-4 were 1.0490 with unlabeled and 1.0364 with deuterated fluoromalate. With APADP, the corresponding values were 1.0138 and 1.0087. These data show that the mechanism is stepwise with both nucleotide substrates, in contrast to the reaction of malate and APADP, which was postulated to be concerted by Karsten et al. [Karsten, W. E., and Cook, P. F. (1994) Biochemistry 33, 2096-2103], a conclusion recently shown to be correct by Edens et al. [Edens, W. A., Urbauer, J. L., and Cleland, W. W. (1997) Biochemistry 36, 1141-1147]. To explain the effect of deuteration on the 13C isotope effect with APADP, it is necessary to assume a secondary 13C isotope effect at C-4 on the hydride transfer step of approximately 1.0064 (assuming 5.7 as the intrinsic primary deuterium isotope effect and 1.054 as the product of the 13C equilibrium isotope effect on hydride transfer and the intrinsic 13C isotope effect on decarboxylation). The secondary 13C isotope effect on hydride transfer is thought to result from hyperconjugation between the carbonyl group and C-4 of the enzyme-bound fluorooxaloacetate intermediate.
3-Fluorooxalacetate is a substrate for malic dehydrogenase. When enzymatic reduction is slower than the rate of epimerization of the two enantiomers, only (2R,3R)-erythro-fluoromalate is formed. Conversely, when a high enzyme level and excess of NADH lead to reduction that is fast relative to the epimerization rate, equal amounts of (2R,3R)-erythro- and (2R,3S)-threo-fluoromalate are formed. These data suggest that the V/K value for reduction of the R enantiomer to give the erythro isomer is approximately 100 times greater than for reduction of the S enantiomer to give the threo isomer. The equilibrium constant for the oxidation of fluoromalate is an order of magnitude less favorable than for oxidation of malate, while the equilibrium deuterium isotope effect from deuteration at C-2 of the substrate is 1.09 for fluoromalate versus 1.18 for malate. These effects reflect the inductive effect of fluorine at the 3-position.
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