The mechanistic pathway for the reaction catalyzed by Kdo8P synthase has been investigated, and the cyclic bisphosphate 2 has been examined as a putative reaction intermediate. Two parallel approaches were used: (1) chemical synthesis of 2 and evaluation as an alternate substrate for the enzyme and (2) transient kinetic studies using rapid chemical quench methodology to provide direct observation and characterization of putative intermediate(s) during enzyme catalysis. The putative cyclic bisphosphate intermediate 2, possessing the stereochemistry of the beta-pyranose form, was synthesized and evaluated as a substrate and as an inhibitor of Kdo8P synthase. The substrate activity was examined by monitoring the release of anomeric phosphate over time using proton-decoupled 31P NMR spectroscopy. A very similar time course for the formation of inorganic phosphate was found in each experiment and the corresponding control experiment; i.e., no enzyme-catalyzed acceleration in the anomeric phosphate hydrolysis was detected. It was found however that 2 binds to the enzyme and is a competitive inhibitor with respect to phosphoenolpyruvate binding, having a Ki value of 35 microM. In a parallel study, we have performed single-turnover rapid chemical quench experiments to examine both the forward and reverse directions to identify a putative enzyme intermediate(s). Our results clearly demonstrate that the cyclic bisphosphate intermediate 2 does not accumulate under single-enzyme turnover conditions. This observation, coupled with the results obtained through the evaluation of synthetic 2 as a substrate, strongly suggests that the Kdo8P synthase catalytic pathway does not involve the formation of 2 as a reaction intermediate. Taken together, these combined results support the original hypothesis [Hedstrom, L., and Abeles, R. H. (1988) Biochem. Biophys. Res. Commun. 157, 816-820], which suggests a reaction pathway involving an acyclic bisphosphate intermediate 1.