The multiple amidation reactions catalyzed by Cobyric acid synthetase from Salmonella typhimurium are sequential and dissociative.

Vitamin B12 is utilized as an essential cofactor in a wide range of enzyme-catalyzed reactions. Most of the enzymes involved in the biosynthesis of this complex macromolecule have now been identified and biochemically characterized.1 Cobyric acid synthetase (CbiP) from Salmonella typhimurium catalyzes the ATP-dependent amidation of adenosyl-cobyrinic acid a,c-diamide (I) at carboxylates b, d, e, and g to produce cobyric acid (II) using glutamine or ammonia as the nitrogen source (Scheme 1). In the amidotransferase family of enzymes the production and utilization of ammonia takes place at physically distinct active sites. The ammonia, generated from the hydrolysis of glutamine, is translocated through the interior of the protein via an intramolecular tunnel to a separate active site for amide bond synthesis.2 The reaction catalyzed by CbiP is particularly intriguing since a single synthetase active site catalyzes the amidation of four different carboxylate groups attached to the periphery of a single corrinoid substrate. It is not known whether the overall reaction mechanism is dissociatiVe or processiVe. It is also unknown whether the four carboxylates are amidated in an ordered or random sequence. In this Communication we have determined the specific sequence of the four consecutive amidation reactions and the relative rates for the appearance and disappearance of the partially amidated intermediates.