Synthesis of (2R,3R)-erythro- and (2R,3S)-threo-Fluoromalate Using Malic Dehydrogenase; Stereoselectivity of Malic Dehydrogenase

Abstract

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.

Mechanisms of Enzymatic Oxidative Decarboxylation

“…So why is the reaction of malate with acetylpyridine-NADP concerted, while reaction of NADP and malate, or reaction of fluoromalate with either nucleotide, is stepwise? The substitution with fluorine makes oxidation of malate more difficult by an order of magnitude, while replacement of NADP with acetylpyridine-NADP makes it more favorable by a factor of 126 . We believe it is the increased equilibrium constant of the overall reaction which converts a stepwise reaction with decarboxylation as the highest barrier to a concerted one.…”

“…The substitution with fluorine makes oxidation of malate more difficult by an order of magnitude, while replacement of NADP with acetylpyridine-NADP makes it more favorable by a factor of 126. 12 We believe it is the increased equilibrium constant of the overall reaction which converts a stepwise reaction with decarboxylation as the highest barrier to a concerted one. Lowering the energy level of E-NADPH-pyruvate relative to E-NADPmalate decreases the barrier to decarboxylation to the point where it disappears and the reaction becomes an asynchronous concerted one.…”

“…With the data for acetylpyridine-NADP, however, eq 1 was not satisfied. With D K eq = 1.09, this equation gave 1.59 for the left side and 2.94 for the right side . To explain this result, it was necessary to assume a secondary 13 C isotope effect on the hydride-transfer step of ∼1.0064.…”