Table 2.
Activation and kinetics of IslA variants from reactions repeated in triplicate
Protein | Percentage of active sites containing glycyl radical (%) | Detectable activity (sulfite) | KM (mM) | kcat (s−1) | Glycyl radical normalized kcat (s−1) | Catalytic efficiency using normalized kcat (s−1 M−1) |
---|---|---|---|---|---|---|
Wild-type | 20.8 ± 0.5 | yes | 8 ± 2 | 2.0 ± 0.1 | 9.5 ± 0.6 | 1200 ± 310 |
G805A | 0 | no | N/D | N/D | N/D | |
C468S | 22.1 ± 0.4 | no | N/D | N/D | N/D | |
E470Q | 11.2 ± 0.7 | no | N/D | N/D | N/D | |
Q193A | 17 ± 1 | no | N/D | N/D | N/D | |
R189E | 28.1 ± 0.6 | no | N/D | N/D | N/D | |
R678E | 5.9 ± 0.1 | no | N/D | N/D | N/D | |
R189E/R678E | 11 ± 1 | no | N/D | N/D | N/D | |
F682A | 11 ± 1 | yes | 6.8 ± 0.6 | 0.0171 ± 0.0004 | 0.154 ± 0.004 | 23 ± 2.1 |
F682Y | 32.2 ± 0.8 | yes | 8 ± 1 | 0.0074 ± 0.0003 | 0.023 ± 0.001 | 2.9 ± 0.4 |
W374F | 47 ± 2 | yes | 11 ± 1 | 0.063 ± 0.003 | 0.133 ± 0.006 | 12 ± 1.2 |
W374Y | 30.2 ± 0.9 | yes | 16.0 ± 0.9 | 0.140 ± 0.003 | 0.465 ± 0.009 | 29 ± 1.7 |
I192A | 21 ± 1 | yes | 12 ± 2 | 0.40 ± 0.02 | 1.9 ± 0.1 | 160 ± 28 |
V680A | 52 ± 3 | no | N/D | N/D | N/D |
Percentage of active sites containing glycyl radical indicate mean ± standard deviations; kinetic parameters are listed as mean ± standard error. N/D, not determined