A base-off analogue of coenzyme-B12 with a modified nucleotide loop--1H-NMR structure analysis and kinetic studies with (R)-methylmalonyl-CoA mutase, glycerol dehydratase, and diol dehydratase.
(Co beta-5'-Deoxyadenosin-5'-yl)-(p-cresolyl)cobamide (Ado-PCC), an analogue of the base-off form of coenzyme-B12 (CoB12), was prepared by alkylation of (Co alpha/beta-cyano/aqua)-(p-cresolyl)cobamide (PCC) with 5'-chloro-5'-deoxyadenosine. The 500 MHz 1H-NMR spectrum of Ado-PCC in D2O at pH 7.4 was completely analyzed using COSY and NOESY two-dimensional experiments. The coenzyme and inhibitory activities of Ado-PCC were tested with three coenzyme-B12-dependent enzymes: (R)-methylmalonyl-CoA mutase, glycerol dehydratase, and diol dehydratase. Ado-PCC showed strong coenzyme activity with methylmalonyl-CoA mutase, which is known to bind the base-off form of CoB12. In contrast, Ado-PCC had no coenzyme activity but acted instead as a competitive inhibitor with glycerol dehydratase and diol dehydratase, which are likely to prefer the base-on form of CoB12. These results indicate that Ado-PCC, whose structure is analogous to the base-off form of CoB12, can be used for probing the mode of coenzyme binding by coenzyme-B12-dependent enzymes.
Poppe
L
L
Department of Biochemistry, Institute of Organic Chemistry, University of Karlsruhe, Germany.
Stupperich
E
E
Hull
W E
WE
Buckel
T
T
Rétey
J
J
D003038:Cobamides / Q000737:chemistry* / Q000378:metabolism; D006836:Hydro-Lyases / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008765:Methylmalonyl-CoA Mutase / Q000378:metabolism*; D009711:Nucleotides / Q000737:chemistry*; D011408:Propanediol Dehydratase / Q000378:metabolism*
D003038:Cobamides; D009711:Nucleotides; D006836:Hydro-Lyases; D011408:Propanediol Dehydratase; C020671:glycerol dehydratase; D008765:Methylmalonyl-CoA Mutase; C000913:cobamamide