Roles of the beta-D-ribofuranose ring and the functional groups of the D-ribose moiety of adenosylcobalamin in the diol dehydratase reaction.

Four analogs of adenosylcobalamin (AdoCbl) modified in the D-ribose moiety of the Co beta ligand were synthesized, and their coenzymic properties were studied with diol dehydratase of Klebsiella pneumoniae ATCC 8724. 2'-Deoxyadenosylcobalamin (2'-dAdoCbl) and 3'-deoxyadenosylcobalamin (3'-dAdoCbl) were active as coenzyme. 2',3'-Secoadenosylcobalamin (2',3'-secoAdoCbl), an analog bearing the same functional groups as AdoCbl but nicked between the 2' and 3' positions in the ribose moiety, and its 2',3'-dialdehyde derivative (2',3'-secoAdoCbl dialdehyde) were totally inactive analogs of the coenzyme. It is therefore evident that the beta-D-ribofuranose ring itself, possibly its rigid structure, is essential and much more important than the functional groups of the ribose moiety for coenzymic function (relative importance: beta-D-ribofuranose ring much greater than 3'-OH greater than 2'-OH greater than ether group). With 2'-dAdoCbl and 3'-dAdoCbl as coenzymes, an absorption peak at 478 nm appeared during enzymatic reaction, suggesting homolysis of the C-Co bond to form cob(II)alamin as intermediate. In the absence of substrate, the complexes of the enzyme with these active analogs underwent rapid inactivation by oxygen. This suggests that their C-Co bond is activated even in the absence of substrate by binding to the apoprotein. No significant spectral changes were observed with 2',3'-secoAdoCbl upon binding to the apoenzyme. In contrast, spectroscopic observation indicates that 2'3'-secoAdoCbl dialdehyde, another inactive analog, underwent gradual and irreversible cleavage of the C-Co bond by interaction with the apodiol dehydratase, forming the enzyme-bound cob(II)alamin without intermediates.