Table 1.
Kinetic Parameters of the Recombinant Proteins
| Substrates | Enzymes | Km (μM) | kcat (s−1) | kcat/Km (M−1·s−1) |
|---|---|---|---|---|
| DTNB | TmGrx1 | 5.15±0.80 | 3.50±0.24 | 6.8×105 |
| TmGrx2 | 0.84±0.08 | 1.21±0.04 | 1.4×106 | |
| TmGrx3 | 11.39±1.55 | 0.66±0.04 | 5.8×104 | |
| Benzoquinone | TmPrxNtr | ≤20 | 52.80±7.20 | ≥9.4×106 |
| TmPrxNtrC40S | ≤20 | 44.10±5.45 | ≥6.3×106 | |
| TmPrxNtr-Ntr2 | ≤20 | 66.80±6.70 | ≥1.2×107 | |
| Chromate | TmPrxNtr | ≤20 | 21±1.34 | ≥3.7×106 |
| TmPrxNtrC40S | ≤20 | 19.80±1.05 | ≥2.8×106 | |
| TmPrxNtr-Ntr2 | ≤20 | 25±1.52 | ≥4.6×106 | |
| H2O2 | TmPrxNtr-Prx | 13.3±4.1 | 0.27±0.01 | 2.0×104 |
| TmPrxBCP | 39.6±4.5 | 0.12±0.01 | 3.1×103 |
All kinetic parameters have been obtained under steady-state conditions. For DTNB and H2O2 reducing activities, the results are expressed as nmol substrates reduced by nmole enzymes per second. For DTNB assay, the Km values represent the affinity of TmTR for the various Grxs-like. Hence, the catalytic efficiency corresponds here to a kcat/KGrx. For benzoquinone and chromate reductase activities, the affinity is so high that the Km cannot be accurately determined with this spectrophotometric assay. The turnover numbers have been expressed as nmol substrate reduced by nmol FMN per second, owing to the fact that the recombinant FMN-containing enzymes (TmPrxNtr, TmPrxNtrC40S, and TmPrxNtr-Ntr2) only contained 0.28, 0.35, and 0.27 mole FMN/mole monomer, respectively.
DTNB, 5,5′-dithio-bis-2-nitrobenzoic acid; FMN, flavin mononucleotide; Grx, glutaredoxin; Nrx, nitroreductase; Prx, peroxiredoxin.