Table 2. Carboxylase mutants with Ala substituted for Cys99 and/or Cys450 retain significant activity.
| Carboxylase variant | Carboxylase specific activity, pmol/ng·h-1 (%) | Epoxidase specific activity, pmol/ng·h-1 (%) |
|---|---|---|
| Single mutants | ||
| Wild type | 26.5 (100) | 27.8 (100) |
| C99A | 8.3 (31) | 9.4 (34) |
| C450A | 8.9 (34) | 11.1 (40) |
| Mock | 0 (0) | 0 (0) |
| Double mutants | ||
| Wild type | 18.0 (100) | 18.7 (100) |
| C99S/C450S | 3.2 (18) | 2.9 (16) |
| C99A/C450A | 8.2 (46) | 7.4 (40) |
| Mock | 0 (0) | 0 (0) |
Microsomes from baculoviral- or mock-infected insect cells were solubilized in the presence of propeptide, and carboxylase specific activities were determined by carboxylase assay (by using a specific activity of 40 cpm/pmol for 14CO2) and quantitative Western blots as described in the legend to Table 1. Aliquots from the same preparations were also assayed on the same day for epoxidase activity. The sets of solubilized microsomes in the single and double mutant groups were analyzed on separate days, which accounts for the difference in the wild-type specific activities.