TABLE 3.
Kinetic parameters of wild-type and class M CobT proteinsa
| Protein | Sp act (μmol/min/mg) | KmNaMN (mM) | kcat (s−1) | kcat/Km(s−1 mM−1) | % Loss of catalytic efficiency |
|---|---|---|---|---|---|
| CobTWT | 30 ± 2 | 0.3 ± 0.03 | 38 ± 2 | 138 ± 22 | 0 |
| CobT(C160A) | 25 ± 2 | 1.2 ± 0.5 | 31 ± 3 | 29 ± 12 | 79 |
| CobT(G257A) | 11 ± 3 | 0.3 ± 0.05 | 14 ± 3 | 50 ± 14 | 64 |
| CobT(G257D) | 0.1 ± 0.01 | 2.2 ± 1.0 | 0.2 ± 0.1 | 0.1 ± 0.1 | >99 |
| CobT(G171D) | 0.5 ± 0.05 | 0.9 ± 0.15 | 0.6 ± 0.06 | 0.7 ± 0.04 | >99 |
| CobT(G320D) | 0.2 ± 0.1 | 1.0 ± 0.1 | 0.2 ± 0.01 | 0.2 ± 0.02 | >99 |
a
The amount of total protein used in the assays was adjusted to a concentration that resulted in less than 10% of product formed. The activities of the CobTWT, CobT(C160A), and CobT(G257A) proteins were assayed by using 2.5 ng of total protein, while the activities of the CobT(G320D), CobT(G171D), and CobT(G257D) proteins were assayed by using 500, 50, and 200 ng of protein, respectively. The activities of the CobTWT, CobT(C160A), CobT(G257A), CobT(G320D), CobT(G171D), and CobT(G257D) proteins were assayed at 5, 5, 12, 10, 15, and 4 min, respectively.