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

Authors


J. Rétey, Lehrstuhl Biochemie am Institut für Organische Chemie der Universität Karlsruhe, Kaiserstraße 12, D-76128 Karlsruhe, Germany
Fax:+49 721 6084823.
E-mail:biochem@ochhades.chemie.uni-karlsruhe.de

Abstract

(Coβ-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 (Coα/β-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.

Abbreviations
CoB12

coenzyme B12

HO-Cbl

aquacobalamin (vitamin B12a)

CN-Cbl

cyanocobalamin (vitamin B12)

PCC

(CN/aq)-p-cresolyl-Cba

Ado-PCC

(CN/aq)-p-cresolyl-AdoCba

Enzymes
 

Glycerol dehydratase (EC 4.2.1.30)

 

propanediol dehydratase (EC 4.2.1.28)

 

methylmalonyl-CoA mutase (EC 5.4.99.2)

 

alcohol dehydrogenase (EC 1.1.1.1)

 

dehydrogenase (EC 1.1.1.37)

 

methylmalonyl-CoA epimerase (EC 5.1.99.1)

 

methylmalonyl-CoA carboxyl-transferase (EC 2.1.3.1)

Ancillary