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A deuterium kinetic isotope effect of 2.1 was observed when [2H3]pyruvate was used as the substrate for pyruvate carboxylase. The effect is on Vmax/Km alone and disappears at infinite substrate concentration. This is interpreted to mean that the slowest step in the overall catalysis is in the half-reaction involving the carboxylation of enzymebiotin by ATP and HCO3-. A tritium intramolecular isotope effect of 4.8 and an intermolecular effect of 1.2 were also observed. The former was interpreted as the isotope effect on the "effective kcat", while the latter the one on V max/Km. With these data, the rate constant for binding of pyruvate was estimated to be 4.5 X 10(6) M-1 min-1, and the deuterium kinetic isotope effect on the catalytic step to be 3.1. Relative values for various rate constants were also obtained. Fluoropyruvate was also shown to be a substrate, reacting six times slower. A deuterium kinetic isotope effect of 1.5 was observed, which remained even at infinite substrate concentration. This is interpreted to mean that the slowest step in the overall catalysis is now the carboxylation of fluoropyruvate.

Stereochemical and product analyses have been studied in our continuing work on the bioprocessing of fluorinated substrate analogues. The hydration pathways of the fumarase-catalyzed reaction on fluorofumarate lead to a product distribution of L-threo-beta-fluoromalate to oxalacetate of 1 to 16. The beta-fluoromalate product has not been previously reported. Oxalacetate formation from the initial product, alpha-fluoromalate, an alpha-fluorohydrin, proceeds by way of a direct nonenzymic decomposition path (as opposed to collapse to the enol of oxalacetate with subsequent tautomerization). Difluorofumarate is hydrated to an alpha-fluorohydrin, alpha, beta-difluoromalate, which decomposes to 3(S)-fluorooxalacetate trapped by in situ malate dehydrogenase mediated reduction to L-threo-beta-fluoromalate (2R,3S). L-threo-Fluoro[2-3H]-malate is a slow substrate for the reverse reaction as measured by labilization of 3H while the erythro isomer is barely detectable. The pathways responsible for this volatilization are discussed. Acetylenedicarboxylate hydration stereochemistry was also determined where the initial product of the reaction, the enol of oxalacetate, tautomerized and was trapped by enzymic reduction to L-malate.

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