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J. Biol. Chem., Vol. 276, Issue 2, 1015-1019, January 12, 2001

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The Coenzyme B₁₂ Analog 5'-Deoxyadenosylcobinamide-GDP Supports Catalysis by Methylmalonyl-CoA Mutase in the Absence of Trans-ligand Coordination^*

Shantanu Chowdhury, Michael G. Thomas^§, Jorge C. Escalante-Semerena^§, and Ruma Banerjee^¶

From the  Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588-0664 and ^§ Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706-1567

Methylmalonyl-CoA mutase is an 5'-adenosylcobalamin (AdoCbl)-dependent enzyme that catalyzes the rearrangement of methylmalonyl-CoA to succinyl-CoA. The crystal structure of this protein revealed that binding of the cofactor is accompanied by a significant conformational change in which dimethylbenzimidazole, the lower axial ligand to cobalt in solution, is replaced by His⁶¹⁰ 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 the subject of enduring debate in the model inorganic literature. In this study, we have used a cofactor analog, 5'deoxyadenosylcobinamide GDP (AdoCbi-GDP), which reconstitutes the enzyme in a "histidine-off" form and which allows 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 factor of 4 compared with the native cofactor AdoCbl. The overall deuterium isotope effect in the presence of AdoCbi-GDP (^DV = 7.2 ± 0.8) is comparable with that observed in the presence of AdoCbl (5.0 ± 0.6) and indicates that the hydrogen transfer steps in this reaction are not significantly affected by the change in coordination state of the bound cofactor. These surprising results are in marked contrast to the effects ascribed to the corresponding lower axial histidine ligands in the cobalamin-dependent enzymes glutamate mutase and methionine synthase.

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				M. Vlasie, S. Chowdhury, and R. Banerjee Importance of the Histidine Ligand to Coenzyme B12 in the Reaction Catalyzed by Methylmalonyl-CoA Mutase J. Biol. Chem., May 17, 2002; 277(21): 18523 - 18527. [Abstract] [Full Text] [PDF]