Determination of the Kinetic and Chemical Mechanism of Malic Enzyme Using (2R,3R)-erythro-Fluoromalate as a Slow Alternate Substrate

Abstract

(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.

Crystal structure of human mitochondrial NAD(P)+-dependent malic enzyme: a new class of oxidative decarboxylases

“…Catalysis by MEs generally proceeds in two steps -the dehydrogenation of malate to produce oxaloacetate (the same reaction as that catalyzed by MDH), and decarboxylation to produce pyruvate [5]. A general base is needed for the first step to extract the proton for dehydrogenation, and a general acid is needed for the second step to protonate the product of decarboxylation.…”

“…Malic enzyme (ME; EC 1.1.1.40) catalyzes the oxidative decarboxylation of L-malate to pyruvate with the concomitant reduction of the cofactor NAD + or NADP + [1][2][3][4][5]. In addition to NAD(P) + , the enzyme also requires divalent cations (Mg 2+ or Mn 2+ ) as cofactors.…”

The Use of Isotope Effects to Determine Enzyme Mechanisms

“…4). Interestingly, Jeff Urbauer showed that with erythrofluoromalate and acetylpyridine-NADP the mechanism remained stepwise, because the fluorine substitution decreases the equilibrium constant for the reaction by an order of magnitude (41).…”

Mechanisms of Enzymatic Oxidative Decarboxylation

“…The expected effects of hyperconjugation in the oxaloacetate intermediate were, however, seen with the slow alternate substrate (2R,3R)-erythro-fluoromalate. 11 This substrate has a turnover number decreased by a factor of 3300, so the chemistry is certainly rate limiting. Deuteration of fluoromalate at C-2 gave a V/K isotope effect of 1.39 with NADP and 3.32 with acetypyridine-NADP.…”