Table 2.
Kinetic parameters of LrPduO wild type and variants using the Co2+ assay. The substrate cob(II)alamin was generated using a protein reducing system
Enzyme | ATP | Cob(II)alamin | ||||
---|---|---|---|---|---|---|
Km (μM) | kcat (s−1) | kcat/Km (M−1 s−1) | Km (μM) | kcat (s−1) | kcat/Km (M−1 s−1) | |
wild type | 5.5 ± 1.1 | (2.9 ± 0.1) × 10−2 | (5.5 ± 1.1) × 103 | 7.8 ± 1.1 | (3.8 ± 0.5) × 10−2 | (4.8 ± 0.9) × 103 |
F112A | UDa | UD | UD | UD | ||
F112H | 10.4 ± 0.8 | (5.9 ± 0.2) × 10−4 | (5.7 ± 0.5) × 101 | 53.0 ± 8.6 | (6.7 ± 0.2) × 10−4 | (1.3 ± 0.2) × 101 |
F112Y | 9.4 ± 0.4 | (2.3 ± 0.1) × 10−3 | (2.5 ± 0.2) × 102 | 34.2 ± 10.7 | (2.7 ± 0.4) × 10−3 | (8.3 ± 2.8) × 101 |
F112W | 7.3 ± 0.1 | (2.8 ± 0.1) × 10−3 | (3.9 ± 0.2) × 102 | 28.8 ± 5.7 | (3.2 ± 0.1) × 10−3 | (1.1 ± 0.2) × 102 |
F163A | 96.1 ± 7.1b | (1.5 ± 0.1) × 10−2 b | (1.6 ± 0.2) × 102 b | 134 ± 13 | (2.7 ± 0.4) ×10−2 | (2.0 ± 0.3) × 102 |
F187A | 7.9 ± 1.0 | (1.4 ± 0.1) × 10−2 | (1.8 ± 0.3) × 103 | 15.8 ± 3 | (1.5 ± 0.1) × 10−2 | (9.3 ± 1.9) × 102 |
ΔS183 | 9.9 ± 0.9 | (1.7 ± 0.1) × 10−2 | (1.7 ± 0.2) × 103 | 16.3 ± 1.7 | (1.8 ± 0.1) × 10−2 | (1.1 ± 0.1) × 103 |
Unable to determine constants due to extremely low activity
Kinetic parameters obtained under sub-saturating concentrations of cob(II)alamin
Saturating ATP was at least 100-fold the KM, however saturating cob(II)alamin was limited to 3- to 10- fold the KM as a result of assay sensitivity.