Elucidation of the coenzyme binding mode of further B-12-dependent enzymesusing a base-off analogue of coenzyme B-12

(Co beta-5'-Deoxyadenosin-5'-yl)-(p-cresyl)cobamide (Ado-PCC), a base-off a nalogue of coenzyme B-12 (Ado-Cbl), was used to elucidate the coenzyme B-12 binding mode of glutamate mutase, 2-methyleneglutarate mutase and ethanola mine ammonia-lyase (EAL). Ado-PCC functions as excellent coenzyme for the c arbon skeleton rearrangements with apparent K-m values of 200 and 10 nM for glutamate and 2-methyleneglutarate mutases, respectively. The correspondin g values for Ado-Cbl are 60 and 54 nM, respectively. In contrast, Ado-PCC s howed no coenzyme activity with EAL but was a competitive inhibitor with re spect to Ado-Cbl. The K-i value for Ado-PCC was 26 nM, the apparent K-m val ue for Ado-Cbl was 30 nM. These results are in agreement with the notion th at in glutamate and 2-methyleneglutarate mutases, a conserved histidine res idue of the protein is coordinated to the cobalt atom of coenzyme B-12, whe reas in EAL the dimethylbenzimidazole residue of the coenzyme itself serves as ligand. (C) 2000 Elsevier Science B.V. All rights reserved.