Abstract
Roles of the components of the nucleotide loop of adenosylcobalamin (AdoCbl) in catalysis were studied with diol dehydrase using synthetic analogs of AdoCbl. Neither the α-D-ribofuranose ring nor the functional groups of the ribose moiety were essential for coenzymic function. Therefore, the D-ribose moiety seems important as a spacer to keep 5,6-dimethylbenzimidazole (DBI) in the proper position. Coenzyme analogs in which the D-ribose and DBI moieties of the coenzyme were replaced by a trimethylene and imidazole or pyridine, respectively, induced suicidal inactivation of enzyme .Adenosylcobinamide methyl phosphate behaved as a pseudocoenzyme, although adenosylcobinamide neither functioned as coenzyme nor bound tightly to apoenzyme. Therefore, it is evident that the phosphodiester moiety of the nucleotide loop is essential for tight binding to apoenzyme and that the DBI moiety of AdoCbl plays a pivotal role in protecting the highly reactive radical intermediates from unfruitful side reactions. Evidence is presented to show that cobalamin is bound to the enzyme with DBI coordinated to the cobalt atom. It is also concluded that the nucleotide loop-modified analogs are useful probes for exploring the cobalamin binding sites of proteins.
