Role of active site residues in promoting cobalt-carbon bond homolysis in adenosylcobalamin-dependent mutases revealed through experiment and computation.

Adenosylcobalamin (AdoCbl) serves as a source of reactive free radicals that are generated by homolytic scission of the coenzyme's cobalt-carbon bond. AdoCbl-dependent enzymes accelerate AdoCbl homolysis by ∼10(12)-fold, but the mechanism by which this is accomplished remains unclear. We have combined experimental and computational approaches to gain molecular-level insight into this process for glutamate mutase. Two residues, glutamate 330 and lysine 326, form hydrogen bonds with the adenosyl group of the coenzyme. A series of mutations that impair the enzyme's ability to catalyze coenzyme homolysis and tritium exchange with the substrate by 2-4 orders of magnitude were introduced at these positions. These mutations, together with the wild-type enzyme, were also characterized in silico by molecular dynamics simulations of the enzyme-AdoCbl-substrate complex with AdoCbl modeled in the associated (Co-C bond formed) or dissociated [adenosyl radical with cob(II)alamin] state. The simulations reveal that the number of hydrogen bonds between the adenosyl group and the protein side chains increases in the homolytically dissociated state, with respect to the associated state, for both the wild-type and mutant enzymes. The mutations also cause a progressive increase in the mean distance between the 5'-carbon of the adenosyl radical and the abstractable hydrogen of the substrate. Interestingly, the distance between the 5'-carbon and substrate hydrogen, determined computationally, was found to inversely correlate with the log k for tritium exchange (r = 0.93) determined experimentally. Taken together, these results point to a dual role for these residues: they both stabilize the homolytic state through electrostatic interactions between the protein and the dissociated coenzyme and correctly position the adenosyl radical to facilitate the abstraction of hydrogen from the substrate.