STRUCTURAL AND ELECTRONIC SIMILARITY BUT FUNCTIONAL DIFFERENCE IN METHYLMALONYL-COA MUTASE BETWEEN COENZYME B-12 AND THE ANALOG 2',5'-DIDEOXYADENOSYLCOBALAMIN

The cofactor analog 2',5'-dideoxyadenosylcobalamin (ddAdoCbl) differs from the natural cofactor coenzyme B-12 [5'-deoxyadenosylcobalamin (dA doCbl)] by lacking only one oxygen atom. The H-1 and C-13 NMR spectra of ddAdoCbl have been assigned unambiguously by homonuclear and hetero nuclear 2D NMR techniques. The H-1, C-13, and P-3l chemical shift valu es for ddAdoCbl were compared with those of another organocobalamin, n amely dAdoCbl. This assessment shows that the analog is very similar b oth electronically and structurally to the natural cofactor. The effec tiveness of ddAdoCbl as a cofactor for both the human and Propionibact erium shermanii methylmalonyl-CoA mutases was compared with that of th e natural cofactor. ddAdoCbl was found to be a competitive inhibitor w ith respect to dAdoCbl. Similar binding affinities to both enzymes wer e found for both the ddAdoCbl analog and the natural cofactor. However , in the presence of ddAdoCbl, the rate of conversion of methylmalonyl CoA to succinyl-CoA was only 1-2% of that seen with the natural cofac tor. There were no changes with time in the visible absorption spectru m of the bound cofactor analog in the presence of substrate, suggestin g that the Co-C bond was not cleaved. The CD (circular dichroism) spec tra of dAdoCbl and ddAdoCbl are very similar, consistent with the NMR results, The CD spectral changes upon binding to P. shermanii methylma lonyl-CoA mutase are large compared to those reported on the binding o f dAdoCbl to ethanolamine ammonia lyase. Furthermore, the CD spectra o f both enzyme-bound cobalamins are very similar, suggesting that simil ar changes in the conformation or structure in these cobalamins occur on binding to the enzyme. Since the natural cofactor binds with the 5, 6-dimethylbenzimidazole displaced by a nitrogenous ligand, probably a His residue, the analog must bind in this same way. The CD spectral ch anges are thus a potential signature for such displacement. The inacti vity of the cofactor most probably does not lie in the nature of the i nteraction of the Co(III) cofactor with the protein, We hypothesize th at the 2'-OH group participates in the formation of the Co(II) form of the cofactor, most likely by stabilization of the initially formed ra dicals.