SYNTHESIS, INHIBITION, AND ACID-CATALYZED HYDROLYSIS STUDIES OF MODELCOMPOUNDS OF THE PROPOSED INTERMEDIATE IN THE KDO8P-SYNTHASE-CATALYZED REACTION

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 alph a-phosphate (compound 6), were synthesized and probed as inhibitors fo r 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 stereoisome rs at the anomeric carbon suggests that the binding subsites of the ph osphate and carboxylate moieties are not absolute in their specificity and may interchange, while the observed one order magnitude preferenc e of the beta-phosphate 5 supports the proposed beta-phosphate configu ration in the putative bisphosphate intermediate. To test the stabilit y of this intermediate structure, the acid-catalyzed phosphate hydroly sis of the analogues 5 and 6 and the carboxylic acid methyl ester 7 we re examined at various pH values, and rate profiles were constructed f rom the data obtained. These data indicate that anomeric carboxylate a ffords catalysis and is more effective than identically oriented carbo xylic acid or ester groups by a factor of at least two orders of magni tude. These kinetic results are inconsistent for two potential rales o f Cl carboxylate function in anomeric phosphate hydrolysis: the intram olecular general acid catalysis and anchimeric assistance,but are cons istent with the inductive stabilizing effect that the carboxylate is e xpected to have on the positive charge being generated on the transien tly formed oxocarbenium ion intermediate in the rate-determining proce ss. The applications of these results to Kdo8P-synthase-catalyzed reac tion are discussed.