D2O of 99.9% isotope purity from Sigma-Aldrich was used to prepare buffers and SAM stocks in D2O. To prepare OphAΔC6-2h in D2O, half of the OphAΔC6-2h proteins concentrated from gel filtration fractions were extensively buffer-exchanged into 25 mM tris buffer prepared in D2O (pD 8.0, where pD = pH* + 0.4, and pH* is the reading number on the pH meter) containing 10% glycerol at room temperature. The other half of OphAΔC6-2h proteins were stored in 25 mM tris (pH 8.0) containing 10% glycerol. Both proteins in H2O and D2O were aliquoted and frozen at −80°C for later experiments. To determine the solvent kinetic isotope effect, parallel reactions containing OphAΔC6-2h (4 mg/ml) and 5 mM SAM were carried out in 50 mM tris buffer (pH 8.0) or in 50 mM tris buffer in D2O (pD 8.0) for 7 hours before being quenched in 4 M urea. To determine the solvent viscosity effect on the reaction, another set of parallel reactions using a different batch of protein was performed in 50 mM tris-HCl (pH 8.0) containing varied concentrations of glycerol (0 and 9%) as the viscosigen. Glycerol (9%) has the same solvent viscosity as D2O. Methylation of individual reactions was analyzed similarly.

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