Stereochemistry of phosphoenolpyruvate carboxylation catalyzed by phosphoenolpyruvate carboxykinase

The stereochemistry of the carboxylation of phosphoenolpyruvate to yield oxalacetate, catalyzed by chicken liver phosphoenolpyruvate carboxykinase and by Ascaris muscle phosphoenolpyruvate carboxykinase, was determined. The substrate (Z)-3-fluorophosphoenolpyruvate was used for the stereochemical analysis. The carboxylation reaction was coupled to malate dehydrogenase to yield 3-fluoromalate, and the stereochemistry of the products was identified by 19F NMR. In separate experiments, the enantiomeric tautomers of 3-fluorooxalacetate were shown to be utilized by malate dehydrogenase to yield (2R,3R)- and (2R,3S)-3-fluoromalate in nearly identical amounts. The products were identified by 19F NMR. When (Z)-3-fluorophosphoenolpyruvate was used as a substrate for phosphoenolpyruvate carboxykinase from avian liver and from Ascaris, and malate dehydrogenase was used to trap the product, only a single diastereomer was observed. This product was shown to be (2R,3R)-3-fluoromalate in each case. The assignments were based on coupling constants taken from Keck et al. [Keck, R., Hess, H., & Rétey, J. (1980) FEBS Lett. 114, 287]. These results indicate that the stereochemistry of carboxylation, catalyzed by chicken phosphoenolpyruvate carboxykinase and by Ascaris phosphoenolpyruvate carboxykinase, is identical and takes place from the si side of the enzyme-bound phosphoenolpyruvate. The carboxylation reaction was run both in H2O and in D2O. No deuterium incorporation into fluoromalate was shown to occur. The product 3-fluorooxalacetate is thus released from phosphoenolpyruvate carboxykinase as the keto form and is reduced more rapidly by reduced nicotinamide adenine dinucleotide with malate dehydrogenase than by the occurrence of tautomerization.