FIGURE 2.
Effect of arginines on the activity and stability of CblC. A, phenylglyoxal inhibits dealkylation of MeCbl by CblC. CblC (30 μm) and MeCbl (20 μm) in Buffer A were incubated with 10 mm GSH and increasing concentrations of phenylglyoxal (0–4 mm) for 30 min, and its effect on the demethylation activity was determined as described under “Experimental Procedures.” Plots were normalized to denote maximum activity in the absence of phenylglyoxal. The data represent the mean ± S.D. of three independent experiments. B, the effect of R161Q/G substitutions on the stability of CblC as monitored at 600 nm. Plots for the temperature-dependent unfolding of R161Q (gray circles) and R161G (open circles) mutants are compared with wild-type (black circles) CblC. The plots were normalized to the starting absorbance at 600 nm representing folded protein. The Tm values were determined as described under “Experimental Procedures.”