Zinc-thiolate intermediate in catalysis of methyl group transfer in Methanosarcina barkeri

S. Gencic, G. M. LeClerc, N. Gorlatova, K. Peariso, J. E. Penner-Hahn, D. A. Grahame

Research output: Contribution to journalArticle

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Abstract

Methyl group transfer reactions are essential in methane-forming pathways in all methanogens. The involvement of zinc in catalysis of methyl group transfer was studied for the methyltransferase enzyme MT2-A important for methanogenesis in Methanosarcina barkeri growing on methylamines. Zinc was shown to be required for MT2-A activity and was tightly bound by the enzyme with an apparent stability constant of 1013.7 at pH 7.2. Oxidation was a factor influencing activity and metal stoichiometry of purified MT2-A preparations. Methods were developed to produce inactive apo MT2-A and to restore full activity with stoichiometric reincorporation of Zn2+. Reconstitution with Co2+ yielded an enzyme with 16-fold higher specific activity. Cysteine thiolate coordination in Co2+-MT2-A was indicated by high absorptivity in the 300-400 nm charge transfer region, consistent with more than one thiolate ligand at the metal center. Approximate tetrahedral geometry was indicated by strong d-d transition absorbance centered at 622 nm. EXAFS analyses of Zn2+-MT2-A revealed 2S + 2N/O coordination with evidence for involvement of histidine. Interaction with the substrate CoM (2-mercaptoethanesulfonic acid) resulted in replacement of the second N/O group with S, indicating direct coordination of the CoM thiolate. UV-visible spectroscopy of Co2+ -MT2-A in the presence of CoM also showed formation of an additional metal-thiolate bond. Binding of CoM over the range of pH 6.2-7.7 obeyed a model in which metalthiolate formation occurs separately from H+ release from the enzyme -substrate complex. Proton release to the solvent takes place from a group with apparent pKa of 6.4, and no evidence for metal-thiolate protonation was found. It was determined that substrate metal-thiolate bond formation occurs with a ΔG° of -6.7 kcal/mol and is a major thermodynamic driving force in the overall process of methyl group transfer.

Original languageEnglish
Pages (from-to)13068-13078
Number of pages11
JournalBiochemistry
Volume40
Issue number43
DOIs
StatePublished - Oct 31 2001
Externally publishedYes

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Methanosarcina barkeri
Catalysis
Zinc
Metals
Methylamines
Substrates
Enzymes
Methanogens
Group Processes
Protonation
Methane
Methyltransferases
Thermodynamics
Histidine
Stoichiometry
Protons
Charge transfer
Spectrum Analysis
Spectroscopy
Ligands

ASJC Scopus subject areas

  • Biochemistry

Cite this

Gencic, S., LeClerc, G. M., Gorlatova, N., Peariso, K., Penner-Hahn, J. E., & Grahame, D. A. (2001). Zinc-thiolate intermediate in catalysis of methyl group transfer in Methanosarcina barkeri. Biochemistry, 40(43), 13068-13078. https://doi.org/10.1021/bi0112917

Zinc-thiolate intermediate in catalysis of methyl group transfer in Methanosarcina barkeri. / Gencic, S.; LeClerc, G. M.; Gorlatova, N.; Peariso, K.; Penner-Hahn, J. E.; Grahame, D. A.

In: Biochemistry, Vol. 40, No. 43, 31.10.2001, p. 13068-13078.

Research output: Contribution to journalArticle

Gencic, S, LeClerc, GM, Gorlatova, N, Peariso, K, Penner-Hahn, JE & Grahame, DA 2001, 'Zinc-thiolate intermediate in catalysis of methyl group transfer in Methanosarcina barkeri', Biochemistry, vol. 40, no. 43, pp. 13068-13078. https://doi.org/10.1021/bi0112917
Gencic S, LeClerc GM, Gorlatova N, Peariso K, Penner-Hahn JE, Grahame DA. Zinc-thiolate intermediate in catalysis of methyl group transfer in Methanosarcina barkeri. Biochemistry. 2001 Oct 31;40(43):13068-13078. https://doi.org/10.1021/bi0112917
Gencic, S. ; LeClerc, G. M. ; Gorlatova, N. ; Peariso, K. ; Penner-Hahn, J. E. ; Grahame, D. A. / Zinc-thiolate intermediate in catalysis of methyl group transfer in Methanosarcina barkeri. In: Biochemistry. 2001 ; Vol. 40, No. 43. pp. 13068-13078.
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abstract = "Methyl group transfer reactions are essential in methane-forming pathways in all methanogens. The involvement of zinc in catalysis of methyl group transfer was studied for the methyltransferase enzyme MT2-A important for methanogenesis in Methanosarcina barkeri growing on methylamines. Zinc was shown to be required for MT2-A activity and was tightly bound by the enzyme with an apparent stability constant of 1013.7 at pH 7.2. Oxidation was a factor influencing activity and metal stoichiometry of purified MT2-A preparations. Methods were developed to produce inactive apo MT2-A and to restore full activity with stoichiometric reincorporation of Zn2+. Reconstitution with Co2+ yielded an enzyme with 16-fold higher specific activity. Cysteine thiolate coordination in Co2+-MT2-A was indicated by high absorptivity in the 300-400 nm charge transfer region, consistent with more than one thiolate ligand at the metal center. Approximate tetrahedral geometry was indicated by strong d-d transition absorbance centered at 622 nm. EXAFS analyses of Zn2+-MT2-A revealed 2S + 2N/O coordination with evidence for involvement of histidine. Interaction with the substrate CoM (2-mercaptoethanesulfonic acid) resulted in replacement of the second N/O group with S, indicating direct coordination of the CoM thiolate. UV-visible spectroscopy of Co2+ -MT2-A in the presence of CoM also showed formation of an additional metal-thiolate bond. Binding of CoM over the range of pH 6.2-7.7 obeyed a model in which metalthiolate formation occurs separately from H+ release from the enzyme -substrate complex. Proton release to the solvent takes place from a group with apparent pKa of 6.4, and no evidence for metal-thiolate protonation was found. It was determined that substrate metal-thiolate bond formation occurs with a ΔG° of -6.7 kcal/mol and is a major thermodynamic driving force in the overall process of methyl group transfer.",
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AU - Gencic, S.

AU - LeClerc, G. M.

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AU - Peariso, K.

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AU - Grahame, D. A.

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N2 - Methyl group transfer reactions are essential in methane-forming pathways in all methanogens. The involvement of zinc in catalysis of methyl group transfer was studied for the methyltransferase enzyme MT2-A important for methanogenesis in Methanosarcina barkeri growing on methylamines. Zinc was shown to be required for MT2-A activity and was tightly bound by the enzyme with an apparent stability constant of 1013.7 at pH 7.2. Oxidation was a factor influencing activity and metal stoichiometry of purified MT2-A preparations. Methods were developed to produce inactive apo MT2-A and to restore full activity with stoichiometric reincorporation of Zn2+. Reconstitution with Co2+ yielded an enzyme with 16-fold higher specific activity. Cysteine thiolate coordination in Co2+-MT2-A was indicated by high absorptivity in the 300-400 nm charge transfer region, consistent with more than one thiolate ligand at the metal center. Approximate tetrahedral geometry was indicated by strong d-d transition absorbance centered at 622 nm. EXAFS analyses of Zn2+-MT2-A revealed 2S + 2N/O coordination with evidence for involvement of histidine. Interaction with the substrate CoM (2-mercaptoethanesulfonic acid) resulted in replacement of the second N/O group with S, indicating direct coordination of the CoM thiolate. UV-visible spectroscopy of Co2+ -MT2-A in the presence of CoM also showed formation of an additional metal-thiolate bond. Binding of CoM over the range of pH 6.2-7.7 obeyed a model in which metalthiolate formation occurs separately from H+ release from the enzyme -substrate complex. Proton release to the solvent takes place from a group with apparent pKa of 6.4, and no evidence for metal-thiolate protonation was found. It was determined that substrate metal-thiolate bond formation occurs with a ΔG° of -6.7 kcal/mol and is a major thermodynamic driving force in the overall process of methyl group transfer.

AB - Methyl group transfer reactions are essential in methane-forming pathways in all methanogens. The involvement of zinc in catalysis of methyl group transfer was studied for the methyltransferase enzyme MT2-A important for methanogenesis in Methanosarcina barkeri growing on methylamines. Zinc was shown to be required for MT2-A activity and was tightly bound by the enzyme with an apparent stability constant of 1013.7 at pH 7.2. Oxidation was a factor influencing activity and metal stoichiometry of purified MT2-A preparations. Methods were developed to produce inactive apo MT2-A and to restore full activity with stoichiometric reincorporation of Zn2+. Reconstitution with Co2+ yielded an enzyme with 16-fold higher specific activity. Cysteine thiolate coordination in Co2+-MT2-A was indicated by high absorptivity in the 300-400 nm charge transfer region, consistent with more than one thiolate ligand at the metal center. Approximate tetrahedral geometry was indicated by strong d-d transition absorbance centered at 622 nm. EXAFS analyses of Zn2+-MT2-A revealed 2S + 2N/O coordination with evidence for involvement of histidine. Interaction with the substrate CoM (2-mercaptoethanesulfonic acid) resulted in replacement of the second N/O group with S, indicating direct coordination of the CoM thiolate. UV-visible spectroscopy of Co2+ -MT2-A in the presence of CoM also showed formation of an additional metal-thiolate bond. Binding of CoM over the range of pH 6.2-7.7 obeyed a model in which metalthiolate formation occurs separately from H+ release from the enzyme -substrate complex. Proton release to the solvent takes place from a group with apparent pKa of 6.4, and no evidence for metal-thiolate protonation was found. It was determined that substrate metal-thiolate bond formation occurs with a ΔG° of -6.7 kcal/mol and is a major thermodynamic driving force in the overall process of methyl group transfer.

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