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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