FIGURE 7.

Activity assay of ObcA. a, total oxalate production was measured using an oxalate kit (Trinity Biotech). The presence of either Obc1* or ObcA failed to generate oxalate, whereas production of oxalate occurred slowly and spontaneously in solution with ObcA, only after continuing the reaction for a long time period (hours to days). b, the reaction profile is displayed for CoA production as a function of time. The decrease in absorbance at 600 nm resulted from 2,6-dichlorophenolindophenol, a dye reacting with the free sulfhydryl group of CoA. Differences in the color code represent reactions in the absence of each component indicated. Initial velocity was determined between 60 and 105 s. c, the metal dependence of ObcA activity was assayed but in the presence of different metal ion. d, the CoA (blue) and total oxalate (red) production were measured using the WT and mutant ObcAs. For the relative activity of the CoA production, the initial velocity was determined as shown in b, and those values were compared with that of the WT ObcA. Total oxalate produced was measured after 5 min of reaction time. In both assays, measurements were performed with 400 μm acetyl-CoA, 1 mm oxaloacetate, 100 μm CoCl₂, 50 nm WT or mutant ObcA, and 800 nm Obc1* and carried out in triplicate for each sample; error bars correspond to the S.E. e, the values for K_m and k_cat obtained by CoA production are listed for the WT ObcA and four mutant enzymes with marginal activity, with S.E. values in parentheses.