The binding site for the adenosyl group of coenzyme B12 in diol dehydrase.
The binding of cob(II)alamin (CblII) and 5'-deoxyadenosine to diol dehydrase was studied spectroscopically and with [U-14C]5'-deoxyadenosine. CblII was bound to this enzyme forming a tight 1:1 complex which was resistant to oxidation by O2 even in the presence of CN-. An irreversible 1:1:1 ternary complex was formed between enzyme, CblII, and 5'-deoxyadenosine, when the enzyme was incubated first with the nucleoside and then with CblII. When this order of addition of the constituents was reversed, no 5'-deoxyadenosine was bound to the enzyme-CblII complex. Hydroxocobalamin could also bind to the enzyme together with the nucleoside, while other cob(III)alamins bearing a bulkier Co beta ligand displaced the nucleoside upon binding to the enzyme. The binding of [U-14C]5'-deoxyadenosine was strongly inhibited by unlabeled 5'-deoxy-ara-adenosine, 4',5'-anhydroadenosine, adenosine, adenine, and 5',8-cyclic adenosine, in this order, but not by 5'-deoxyuridine. These results constitute direct evidence for the presence of the binding site for the adenosyl group of adenosylcobalamin, which is spatially limited to and highly specific for adenine nucleosides. The binding of 5'-deoxyadenosine to the apoenzyme was reversible.
D001665:Binding Sites; D001667:Binding, Competitive; D002250:Carbon Radioisotopes; D017338:Cladribine; D003839:Deoxyadenosines / Q000031:analogs & derivatives / Q000378:metabolism; D006836:Hydro-Lyases / Q000378:metabolism*; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology; D009705:Nucleosides / Q000494:pharmacology; D011408:Propanediol Dehydratase / Q000378:metabolism*; D011485:Protein Binding; D013329:Structure-Activity Relationship; D014805:Vitamin B 12 / Q000378:metabolism*
D002250:Carbon Radioisotopes; D003839:Deoxyadenosines; D009705:Nucleosides; D017338:Cladribine; C044739:5'-chloro-5'-deoxyadenosine; D006836:Hydro-Lyases; D011408:Propanediol Dehydratase; D014805:Vitamin B 12