CATALYTIC MECHANISM OF 3-DEOXY-D-MANNO-2-OCTULOSONATE-8-PHOSPHATE SYNTHASE - THE USE OF SYNTHETIC ANALOGS TO PROBE THE STRUCTURE OF THE PUTATIVE REACTION INTERMEDIATE

The proposed mechanistic pathway for the reaction catalyzed by 3-deoxy -D-manno-2-octulosonate-8-phosphate (Kdo8P) synthase was examined in t erms of the structure of the putative bisphosphate intermediate. Two 2 -deoxy analogues of the product Kdo8P, having been structurally prohib ited from undergoing the ring-opening and possessing the stereochemist ry of either the alpha-pyronase (compound 1) or the beta-pyranose form (compound 2) of the product, were synthesized and probed as inhibitor s for the synthase. It was found that both analogues bind to the enzym e and are competitive inhibitors with respect to phosphoenolpyruvate b inding, having K(i) values of 470 muM and 303 muM, respectively. Compa rison of this data to the K(i) value of the tautomeric mixture of the product Kdo8P (K(i) = 590 muM) suggests that both the alpha- and the b eta-pyranose anomers (65.8% and 3.1%, respectively at neutral pH) bind to the enzyme with a slight (1.13 kJ/mol) preference for the beta-ano mer, and that the C2 hydroxyl does not contribute to the binding. This uncertain stereochemical preference exhibited by the enzyme for the s tereoisomers at the anomeric carbon suggests that the carboxylate bind ing site of the product is indistinct, while the hydroxyl and carboxyl ate binding sites may be interchangeable. More importantly, however, t he isosteric phosphonate analogue phonylmethyl-8-phosphate-D-glycero-D -talo-octonate (3), which mimics the topological and electrostatic pro perties of the proposed cyclic intermediate, was found to be the most potent inhibitor of the enzyme with a K(i) value of 5 muM. Two hithert o unrecognized aspects of the mechanism of the synthase were identifie d. First, the results showing that the cyclic analogues 1, 2 and 3 are inhibitors of the enzyme whereas the previously reported acyclic anal ogue, which contains no carbonyl group at C2 and may thus resemble the open-chain form of Kdo8P, is not an inhibitor, suggest that the pyran ose form and not the open-chain acyclic form of the putative bisphosph ate intermediate is handled by the enzyme. Second, since the overall s tereochemical course of the transformation mediated by the synthase ha s been shown to involve si face addition of phosphoenol-pyruvate to th e re face of the carbonyl of arabinose 5-phosphate, the present observ ation involving analogue 3 suggests that the bisphosphate intermediate formed during the initial steps of synthesis may have the pyranose st ructure with the anomeric phosphate located in the beta-configuration.