Table 1.
Protein | kcat (min−1) | Km (HED) (mM) | kcat/Km (mM−1 min−1) |
---|---|---|---|
cGrx1 | 87 ± 15 | 1.5 ± 0.4 | 58 |
cGrx1/U13C | 0.5 ± 0.1 | 1.5 ± 0.8 | 0.3 |
cGrx1/U13W | ND | - | - |
cGrx1/C16S | 83 ± 8 | 0.9 ± 0.2 | 92 |
cGrx2 | 186 ± 28 | 17.6 ± 5.6 | 11 |
cGrx2/C15S | 396 ± 67 | 18.1 ± 4.5 | 22 |
eGrx1 | 126 ± 8 | 11.2 ± 2.2 | 11 |
Purified proteins were assayed in the standard Grx reaction using HED as a substrate. Km and kcat values were determined by fitting the experimental data to the Michaelis-Menten equation. The protein concentrations of selenoprotein Grxs (cGrx1 and cGrx1/C16S) were normalized by subtracting the concentrations of Trp-inserted forms and subjected to calculating kcat values. cGrx1, wild-type selenoprotein Clostridium Grx1; cGrx1/U13C, Sec-to-Cys mutant of cGrx1; cGrx1/U13W, Sec-to-Trp mutant of cGrx1; cGrx1/C16S, monoselenol mutant of cGrx1; cGrx2, wild-type Cys-containing Clostridium Grx2; cGrx2/C15S, monothiol mutant of cGrx2; eGrx1, E. coli Grx1. ND, activity not detectable.