The stereochemistry of CO(2) addition to phosphoenolpyruvate (PEP) to yield oxaloacetate catalyzed by ATP-dependent Saccharomyces cerevisiae and Anaerobiospirillum succiniciproducens PEP carboxykinases was determined using (Z)-3-fluorophosphoenolpyruvate ((Z)-F-PEP) as a substrate analog. A. succiniciproducens and S. cerevisiae PEP carboxykinases utilized (Z)-F-PEP with 1/14 and 1/47 the respective K(m) values for PEP. On the other hand, in the bacterial and yeast enzymes k(cat) was reduced to 1/67 and 1/48 the value with PEP, respectively. The binding affinity of pyridoxylphosphate-labeled S. cerevisiae and A. succiniciproducens PEP carboxykinases for PEP and (Z)-F-PEP was checked and found to be of similar magnitude for both substrates, suggesting that the lowered K(m) values for the fluorine-containing PEP analog are due to kinetic effects. The lowered k(cat) values when using (Z)-F-PEP as substrate suggest that the electron withdrawing effect of fluorine affects the nucleophilic attack of the double bond of (Z)-F-PEP to CO(2). For the stereochemical analyses, the carboxylation of (Z)-F-PEP was coupled to malate dehydrogenase to yield 3-fluoromalate, which was analyzed by (19)F NMR. The fluoromalate obtained was identified as (2R, 3R)-3-fluoromalate for both the A. succiniciproducens and S. cerevisiae PEP carboxykinases, thus indicating that CO(2) addition to (Z)-F-PEP, and hence PEP, takes place through the 2-si face of the double bond. These results, together with previously published data [Rose, I.A. et al. J. Biol. Chem. 244 (1969) 6130-6133; Hwang, S.H. and Nowak, T. Biochemistry 25 (1986) 5590-5595] indicate that PEP carboxykinases, no matter their nucleotide specificity, catalyze the carboxylation of PEP from the 2-si face of the double bond.