Interactions between coenzyme B₁₂ analogs and adenosylcobalamin-dependent glutamate mutase from Clostridium tetanomorphum

• Published in: The FEBS journal 2008-12, Vol.275 (23), p.5960-5968
• Main Authors: Chen, Hao-Ping, Hsu, Huei-Ju, Hsu, Fang-Ciao, Lai, Chien-Chen, Hsu, Chung-Hua
• Format: Article
• Language: English
• Subjects: adenosylcobalamin; adenosylcobinamide; AdoCbi-GDP; B; B12; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Binding sites; Biochemistry; Catalysis; Chemical synthesis; Clostridium tetanomorphum - enzymology; Cobamides - chemical synthesis; Cobamides - chemistry; Cobamides - metabolism; Dialysis; Enzymes; glutamate mutase; Intramolecular Transferases - chemistry; Intramolecular Transferases - metabolism; Kinetics; Magnetic Resonance Spectroscopy; Molecular Structure; Protein Binding; Ratios; Spectrophotometry; Spectrophotometry, Ultraviolet; Thermodynamics
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/j.1742-4658.2008.06724.x

Adenosylcobalamin (AdoCbl)-dependent glutamate mutase from Clostridium tetanomorphum comprises two weakly-associating subunits, MutS and MutE, which combine with AdoCbl to form the active holo-enzyme. Three coenzyme analogs, methylcobinamide (MeCbi), adenosylcobinamide (AdoCbi) and adeosylcobinamide-GDP (AdoCbi-GDP), were synthesized at milligram scale. Equilibrium dialysis was used to measure the binding of coenzyme B₁₂ analogs to glutamate mutase. Our results show that, unlike AdoCbl-dependent methylmalonyl CoA mutase, the ratio kcat/Km decreased approximately 10⁴-fold in both cases when AdoCbi or AdoCbi-GDP was used as the cofactor. The coenzyme analog-binding studies show that, in the absence of the ribonucleotide tail of AdoCbl, the enzyme's active site cannot correctly accommodate the coenzyme analog AdoCbi. The results presented here shed some light on the cobalt-carbon cleavage mechanism of B₁₂.