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

L. Poppe, Erhard Stupperich, William E. Hull, Thomas Buckel, János Rétey

Research output: Contribution to journalArticle

26 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)303-307
Number of pages5
JournalEuropean Journal of Biochemistry
Volume250
Issue number2
Publication statusPublished - 1997

Fingerprint

glycerol dehydratase
Propanediol Dehydratase
Cobamides
Methylmalonyl-CoA Mutase
Nucleotides
Nuclear magnetic resonance
Kinetics
Coenzymes
cobamamide
Proton Magnetic Resonance Spectroscopy
Alkylation
Enzymes

Keywords

  • (Coβ-5'-deoxyadenosin-5'-yl)-(p-cresolyl)cobamide
  • (R)-methylmalonyl-CoA mutase (propionibacterium shermanii)
  • Base-off analog of coenzyme-B
  • Diol dehydratase (Salmonella typhimurium gene overexpressed in E. coli)
  • Glycerol dehydratase (Citrobacter freundii gene overexpressed in Escherichia coli)

ASJC Scopus subject areas

  • Biochemistry

Cite this

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. / Poppe, L.; Stupperich, Erhard; Hull, William E.; Buckel, Thomas; Rétey, János.

In: European Journal of Biochemistry, Vol. 250, No. 2, 1997, p. 303-307.

Research output: Contribution to journalArticle

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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.",
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AB - (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.

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