Structural and Electronic Similarity but Functional Difference in Methylmalonyl-CoA Mutase between Coenzyme B12 and the Analog 2’,5'-Dideoxyadenosylcobalamin

Antonia M. Calafat, Shinichi Taoka, James M. Puckett, Craig L Semerad, Hon Yan, Laibin Luo, Huilan Chen, Ruma Banerjee, Luigi G. Marzilli

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

The cofactor analog 2,,5'-dideoxyadenosylcobalamin (ddAdoCbl) differs from the natural cofactor coenzyme B12 [S'-deoxyadenosylcobalamin (dAdoCbl)] by lacking only one oxygen atom. The and 13C NMR spectra of ddAdoCbl have been assigned unambiguously by homonuclear and heteronuclear 2D NMR techniques. The 1H, 13C, and 31P chemical shift values for ddAdoCbl were compared with those of another organocobalamin, namely dAdoCbl. This assessment shows that the analog is very similar both electronically and structurally to the natural cofactor. The effectiveness of ddAdoCbl as a cofactor for both the human and Propionibacterium shermanii methylmalonyl-CoA mutases was compared with that of the natural cofactor. ddAdoCbl was found to be a competitive inhibitor with respect to dAdoCbl. Similar binding affinities to both enzymes were found for both the ddAdoCbl analog and the natural cofactor. However, in the presence of ddAdoCbl, the rate of conversion of methylmalonyl- CoA to succinyl-CoA was only 1-2% of that seen with the natural cofactor. There were no changes with time in the visible absorption spectrum of the bound cofactor analog in the presence of substrate, suggesting that the Co-C bond was not cleaved. The CD (circular dichroism) spectra of dAdoCbl and ddAdoCbl are very similar, consistent with the NMR results. The CD spectral changes upon binding to P. shermanii methylmalonyl-CoA mutase are large compared to those reported on the binding of dAdoCbl to ethanolamine ammonia lyase. Furthermore, the CD spectra of both enzyme-bound cobalamins are very similar, suggesting that similar changes in the conformation or structure in these cobalamins occur on binding to the enzyme. Since the natural cofactor binds with the 5,6-dimethylbenzimidazole displaced by a nitrogenous ligand, probably a His residue, the analog must bind in this same way. The CD spectral changes are thus a potential signature for such displacement. The inactivity of the cofactor most probably does not lie in the nature of the interaction of the Co(III) cofactor with the protein. We hypothesize that the 2'-OH group participates in the formation of the Co(II) form of the cofactor, most likely by stabilization of the initially formed radicals.

Original languageEnglish (US)
Pages (from-to)14125-14130
Number of pages6
JournalBiochemistry
Volume34
Issue number43
DOIs
StatePublished - Jan 1 1995

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Methylmalonyl-CoA Mutase
Dichroism
Circular Dichroism
Nuclear magnetic resonance
Vitamin B 12
Ethanolamine Ammonia-Lyase
Enzymes
Propionibacterium
Biomolecular Nuclear Magnetic Resonance
2',5'-dideoxyadenosylcobalamin
cobamamide
Chemical shift
Conformations
Absorption spectra
Stabilization
Oxygen
Ligands

ASJC Scopus subject areas

  • Biochemistry

Cite this

Structural and Electronic Similarity but Functional Difference in Methylmalonyl-CoA Mutase between Coenzyme B12 and the Analog 2’,5'-Dideoxyadenosylcobalamin. / Calafat, Antonia M.; Taoka, Shinichi; Puckett, James M.; Semerad, Craig L; Yan, Hon; Luo, Laibin; Chen, Huilan; Banerjee, Ruma; Marzilli, Luigi G.

In: Biochemistry, Vol. 34, No. 43, 01.01.1995, p. 14125-14130.

Research output: Contribution to journalArticle

Calafat, Antonia M. ; Taoka, Shinichi ; Puckett, James M. ; Semerad, Craig L ; Yan, Hon ; Luo, Laibin ; Chen, Huilan ; Banerjee, Ruma ; Marzilli, Luigi G. / Structural and Electronic Similarity but Functional Difference in Methylmalonyl-CoA Mutase between Coenzyme B12 and the Analog 2’,5'-Dideoxyadenosylcobalamin. In: Biochemistry. 1995 ; Vol. 34, No. 43. pp. 14125-14130.
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abstract = "The cofactor analog 2,,5'-dideoxyadenosylcobalamin (ddAdoCbl) differs from the natural cofactor coenzyme B12 [S'-deoxyadenosylcobalamin (dAdoCbl)] by lacking only one oxygen atom. The and 13C NMR spectra of ddAdoCbl have been assigned unambiguously by homonuclear and heteronuclear 2D NMR techniques. The 1H, 13C, and 31P chemical shift values for ddAdoCbl were compared with those of another organocobalamin, namely dAdoCbl. This assessment shows that the analog is very similar both electronically and structurally to the natural cofactor. The effectiveness of ddAdoCbl as a cofactor for both the human and Propionibacterium shermanii methylmalonyl-CoA mutases was compared with that of the natural cofactor. ddAdoCbl was found to be a competitive inhibitor with respect to dAdoCbl. Similar binding affinities to both enzymes were found for both the ddAdoCbl analog and the natural cofactor. However, in the presence of ddAdoCbl, the rate of conversion of methylmalonyl- CoA to succinyl-CoA was only 1-2{\%} of that seen with the natural cofactor. There were no changes with time in the visible absorption spectrum of the bound cofactor analog in the presence of substrate, suggesting that the Co-C bond was not cleaved. The CD (circular dichroism) spectra of dAdoCbl and ddAdoCbl are very similar, consistent with the NMR results. The CD spectral changes upon binding to P. shermanii methylmalonyl-CoA mutase are large compared to those reported on the binding of dAdoCbl to ethanolamine ammonia lyase. Furthermore, the CD spectra of both enzyme-bound cobalamins are very similar, suggesting that similar changes in the conformation or structure in these cobalamins occur on binding to the enzyme. Since the natural cofactor binds with the 5,6-dimethylbenzimidazole displaced by a nitrogenous ligand, probably a His residue, the analog must bind in this same way. The CD spectral changes are thus a potential signature for such displacement. The inactivity of the cofactor most probably does not lie in the nature of the interaction of the Co(III) cofactor with the protein. We hypothesize that the 2'-OH group participates in the formation of the Co(II) form of the cofactor, most likely by stabilization of the initially formed radicals.",
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AU - Calafat, Antonia M.

AU - Taoka, Shinichi

AU - Puckett, James M.

AU - Semerad, Craig L

AU - Yan, Hon

AU - Luo, Laibin

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AU - Marzilli, Luigi G.

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N2 - The cofactor analog 2,,5'-dideoxyadenosylcobalamin (ddAdoCbl) differs from the natural cofactor coenzyme B12 [S'-deoxyadenosylcobalamin (dAdoCbl)] by lacking only one oxygen atom. The and 13C NMR spectra of ddAdoCbl have been assigned unambiguously by homonuclear and heteronuclear 2D NMR techniques. The 1H, 13C, and 31P chemical shift values for ddAdoCbl were compared with those of another organocobalamin, namely dAdoCbl. This assessment shows that the analog is very similar both electronically and structurally to the natural cofactor. The effectiveness of ddAdoCbl as a cofactor for both the human and Propionibacterium shermanii methylmalonyl-CoA mutases was compared with that of the natural cofactor. ddAdoCbl was found to be a competitive inhibitor with respect to dAdoCbl. Similar binding affinities to both enzymes were found for both the ddAdoCbl analog and the natural cofactor. However, in the presence of ddAdoCbl, the rate of conversion of methylmalonyl- CoA to succinyl-CoA was only 1-2% of that seen with the natural cofactor. There were no changes with time in the visible absorption spectrum of the bound cofactor analog in the presence of substrate, suggesting that the Co-C bond was not cleaved. The CD (circular dichroism) spectra of dAdoCbl and ddAdoCbl are very similar, consistent with the NMR results. The CD spectral changes upon binding to P. shermanii methylmalonyl-CoA mutase are large compared to those reported on the binding of dAdoCbl to ethanolamine ammonia lyase. Furthermore, the CD spectra of both enzyme-bound cobalamins are very similar, suggesting that similar changes in the conformation or structure in these cobalamins occur on binding to the enzyme. Since the natural cofactor binds with the 5,6-dimethylbenzimidazole displaced by a nitrogenous ligand, probably a His residue, the analog must bind in this same way. The CD spectral changes are thus a potential signature for such displacement. The inactivity of the cofactor most probably does not lie in the nature of the interaction of the Co(III) cofactor with the protein. We hypothesize that the 2'-OH group participates in the formation of the Co(II) form of the cofactor, most likely by stabilization of the initially formed radicals.

AB - The cofactor analog 2,,5'-dideoxyadenosylcobalamin (ddAdoCbl) differs from the natural cofactor coenzyme B12 [S'-deoxyadenosylcobalamin (dAdoCbl)] by lacking only one oxygen atom. The and 13C NMR spectra of ddAdoCbl have been assigned unambiguously by homonuclear and heteronuclear 2D NMR techniques. The 1H, 13C, and 31P chemical shift values for ddAdoCbl were compared with those of another organocobalamin, namely dAdoCbl. This assessment shows that the analog is very similar both electronically and structurally to the natural cofactor. The effectiveness of ddAdoCbl as a cofactor for both the human and Propionibacterium shermanii methylmalonyl-CoA mutases was compared with that of the natural cofactor. ddAdoCbl was found to be a competitive inhibitor with respect to dAdoCbl. Similar binding affinities to both enzymes were found for both the ddAdoCbl analog and the natural cofactor. However, in the presence of ddAdoCbl, the rate of conversion of methylmalonyl- CoA to succinyl-CoA was only 1-2% of that seen with the natural cofactor. There were no changes with time in the visible absorption spectrum of the bound cofactor analog in the presence of substrate, suggesting that the Co-C bond was not cleaved. The CD (circular dichroism) spectra of dAdoCbl and ddAdoCbl are very similar, consistent with the NMR results. The CD spectral changes upon binding to P. shermanii methylmalonyl-CoA mutase are large compared to those reported on the binding of dAdoCbl to ethanolamine ammonia lyase. Furthermore, the CD spectra of both enzyme-bound cobalamins are very similar, suggesting that similar changes in the conformation or structure in these cobalamins occur on binding to the enzyme. Since the natural cofactor binds with the 5,6-dimethylbenzimidazole displaced by a nitrogenous ligand, probably a His residue, the analog must bind in this same way. The CD spectral changes are thus a potential signature for such displacement. The inactivity of the cofactor most probably does not lie in the nature of the interaction of the Co(III) cofactor with the protein. We hypothesize that the 2'-OH group participates in the formation of the Co(II) form of the cofactor, most likely by stabilization of the initially formed radicals.

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