Elucidation of the coenzyme binding mode of further B12-dependent enzymes using a base-off analogue of coenzyme B12

(Coβ-5′-Deoxyadenosin-5′-yl)-(p-cresyl)cobamide (Ado-PCC), a base-off analogue of coenzyme B12 (Ado-Cbl), was used to elucidate the coenzyme B12 binding mode of glutamate mutase, 2-methyleneglutarate mutase and ethanolamine ammonia–lyase (EAL). Ado-PCC functions as excellent coenzyme for the carbon skeleton rearrangements with apparent Km values of 200 and 10 nM for glutamate and 2-methyleneglutarate mutases, respectively. The corresponding values for Ado-Cbl are 60 and 54 nM, respectively. In contrast, Ado-PCC showed no coenzyme activity with EAL but was a competitive inhibitor with respect to Ado-Cbl. The Ki value for Ado-PCC was 26 nM, the apparent Km value for Ado-Cbl was 30 nM. These results are in agreement with the notion that in glutamate and 2-methyleneglutarate mutases, a conserved histidine residue of the protein is coordinated to the cobalt atom of coenzyme B12, whereas in EAL the dimethylbenzimidazole residue of the coenzyme itself serves as ligand.