Fig 3.
Reduction of cobamide bound to CmtA and methylation with CH3-THMPT or DMS. The reaction mixture (80-μl final volume) contained 10 μM (final concentration) CmtA reconstituted with aquocobalamin in 50 mM MOPS-KOH buffer (pH 7.0). Reactions were performed at 21°C. At the time indicated by the arrow labeled a, 2.5 μl of 32 mM Ti(III)-citrate (1.0 mM final concentration) was added to reduce aquocobalamin. Additions of methyl donors were made at time points indicated by the arrows: b and e, 1.0 nmol each of CH3-THMPT and DMS, respectively; c and d, 2.0 nmol of CH3-THMPT; f and g, 2.0 nmol of DMS. Absorbencies were corrected for dilution when adding reagents. Symbols: Δ, change in absorbance on addition of CH3-THMPT; ▲, change in absorbance on addition of DMS.