The proposed mechanistic pathway for the reaction catalyzed by 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase was examined in terms of structure and stability of the putative bisphosphate intermediate. Two simplified analogues of the proposed intermediate (3), possessing the stereochemistry of either the beta-phosphate (compound 5) or the alpha-phosphate (compound 6), were synthesized and probed as inhibitors for the enzyme. It was found that both analogues bind to the enzyme and are competitive inhibitors with respect to phosphoenolpyruvate binding, having K-i values of 160 and 1300 mu M, respectively. This uncertain stereochemical preference exhibited by the enzyme for the stereoisomers at the anomeric carbon suggests that the binding subsites of the phosphate and carboxylate moieties are not absolute in their specificity and may interchange, while the observed one order magnitude preference of the beta-phosphate 5 supports the proposed beta-phosphate configuration in the putative bisphosphate intermediate. To test the stability of this intermediate structure, the acid-catalyzed phosphate hydrolysis of the analogues 5 and 6 and the carboxylic acid methyl ester 7 were examined at various pH values, and rate profiles were constructed from the data obtained. These data indicate that anomeric carboxylate affords catalysis and is more effective than identically oriented carboxylic acid or ester groups by a factor of at least two orders of magnitude. These kinetic results are inconsistent for two potential rales of Cl carboxylate function in anomeric phosphate hydrolysis : the intramolecular general acid catalysis and anchimeric assistance,but are consistent with the inductive stabilizing effect that the carboxylate is expected to have on the positive charge being generated on the transiently formed oxocarbenium ion intermediate in the rate-determining process. The applications of these results to Kdo8P-synthase-catalyzed reaction are discussed.