The coenzyme B-12 analog 5 '-deoxyadenosylcobinamide-GDP supports catalysis by methylmalonyl-CoA mutase in the absence of trans-ligand coordination

Methylmalonyl-CoA mutase is an 5'-adenosylcobalamin (AdoCbl)-dependent enzy me that catalyzes the rearrangement of methylmalonyl-CoA to succinyl-CoA Th e crystal structure of this protein revealed that binding of the cofactor i s accompanied by a significant conformational change in which dimethylbenzi midazole, the lower axial ligand to cobalt in solution, is replaced by His( 610) donated by the active site. The role of the lower axial ligand in the trillion fold labilization of the upper axial cobalt-carbon bond has been t he subject of enduring debate in the model inorganic literature. In this st udy, we have used a cofactor analog, 5'deoxyadenosylcobinamide GDP (AdoCbi- GDP), which reconstitutes the enzyme in a "histidine-off' form and which al lows us to evaluate the contribution of the lower axial ligand to catalysis , The k(cat) for the enzyme in the presence of AdoCbi-GDP is reduced by a f actor of 4 compared with the native cofactor AdoCbl, The overall deuterium isotope effect in the presence of AdoCbi-GDP (V-D = 7.2 +/- 0.8) is compara ble with that observed in the presence of AdoCbl (5.0 +/- 0.6) and indicate s that the hydrogen transfer steps in this reaction are not significantly a ffected by the change in coordination state of the bound cofactor. These su rprising results are in marked contrast to the effects ascribed to the corr esponding lower axial histidine ligands in the cobalamin-dependent enzymes glutamate mutase and methionine synthase.