Poppe, László;
Stupperich, Erhard;
Hull, William E.;
Buckel, Thomas;
Rétey, János
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
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Medientyp:
E-Artikel
Titel:
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
:
<sup>1</sup>H‐NMR Structure Analysis and Kinetic Studies with (<i>R</i>)‐Methylmalonyl‐CoA Mutase, Glycerol Dehydratase, and Diol Dehydratase
Beteiligte:
Poppe, László;
Stupperich, Erhard;
Hull, William E.;
Buckel, Thomas;
Rétey, János
Beschreibung:
<jats:p>(Coβ‐5′‐Deoxyadenosin‐5′‐yl)‐(<jats:italic>p</jats:italic>‐cresolyl)cobamide (Ado‐PCC), an analogue of the base‐off form of coenzyme‐B<jats:sub>12</jats:sub> (CoB<jats:sub>12</jats:sub>), was prepared by alkylation of (Coα/β‐cyano/aqua)‐(<jats:italic>p</jats:italic>‐cresolyl)cobamide (PCC) with 5′‐chloro‐5′‐deoxyadenosine. The 500 MHz <jats:sup>1</jats:sup>H‐NMR spectrum of Ado‐PCC in D<jats:sub>2</jats:sub>O 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‐B<jats:sub>12</jats:sub>‐dependent enzymes: (<jats:italic>R</jats:italic>)‐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 CoB<jats:sub>12</jats:sub>. 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 CoB<jats:sub>12</jats:sub>. These results indicate that Ado‐PCC, whose structure is analogous to the base‐off form of CoB<jats:sub>12</jats:sub>, can be used for probing the mode of coenzyme binding by coenzyme‐B<jats:sub>12</jats:sub>‐dependent enzymes.</jats:p>