Mechanisms of carbonyl participation in phosphate ester hydrolysis and their relationship to mechanisms for the carboxylation of biotin

Article abstract of DOI:10.1021/ja00003a038

The enzymic reaction of biotin with bicarbonate and ATP produces N-carboxybiotin, ADP, and phosphate. The intermediates in the reaction are unknown. It has been proposed that the intramolecular reaction of the carbonyl hydrate of methylacetoin diethyl phosphate (1) may serve as a model for the latter part of such a mechanism. In basic solution, 1 reacts rapidly to give two sets of products that can be accounted for by a mechanism involving addition of a hydroxyl group of the carbonyl hydrate to the adjacent phosphate. The rapid reaction of the nonenolizable ester with base rules out earlier mechanistic proposals of the reaction involving an enolization pathway. The course of incorporation of water containing H₂¹⁸O into phosphate ester products was determined by ³¹P NMR analysis. This reveals that the reversion of the carbonyl hydrate to the ketone occurs at a rate which is competitive with reaction at the phosphate ester since the isotopic enrichment of the phosphate group is less than the content of the solvent (76%). In weakly basic solutions a single set of products is obtained, but in more highly alkaline solutions a second set of products develops. The results are consistent with a competition between a mechanism in which the carbonyl hydrate adduct expels phosphate and one in which ethoxide is expelled to give a cyclic phosphate. Reactions of acetoin diethyl phosphate (2) and acetol diethyl phosphate (3) show a larger degree of solvent incorporation and a smaller variation in product distribution, consistent with expectations based on steric and inductive effects. It is concluded that the reaction mechanism parallels that which was proposed for the carboxylation of biotin.