The multiple amidation reactions catalyzed by Cobyric acid synthetase from Salmonella typhimurium are sequential and dissociative.
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abstract
The biosynthesis of coenzyme B12 is a complex process involving more than two dozen enzymatic reactions. Near the end of this biosynthetic pathway cobyric acid synthetase (CbiP) catalyzes the remarkable amidation of four separate carboxylate groups within a single substrate, adenosyl-cobyrinic acid a,c-diamide. The time course for the multiple amidation reactions demonstrates that the partially amidated products are released from the active site after every round of catalysis, and thus the mechanism of the reaction is dissociative. The partially amidated intermediates were shown to be single chemical entities demonstrating that the four carboxylate groups are amidated in a specific reaction sequence. NMR spectroscopy was used to establish that carboxylate e was the first to be amidated followed in turn by d, b, and g. These results indicate that the initial substrate can productively bind to the enzyme active site in only one of four possible orientations. After the amidation of carboxylate e, the first partially amidated intermediate must dissociate from the active site and rebind in an orientation that is rotated by approximately 90. Similar events must therefore follow after the amidation of carboxylates d and b. The structural basis for the dissociative and sequential reaction mechanism coupled with the rigid regiochemistry is unknown. Copyright 2006 American Chemical Society.
