TABLE 2.
Substrate, solvent, and multiple KIE on AAO steady-state constants for p-anisyl alcohol and 2,4-hexadien-1-ol oxidation
Assays were performed in normal and deuterated 100 mm phosphate, pH 6 (at 25 °C, under air saturation conditions) using the α-protiated and α-bideuterated alcohol substrates. Different KIE estimated: Dk, substrate KIE in water (i.e. k for α-protiated substrate in water/k for α-bideuterated substrate in water); Dk(D2O), substrate KIE in D2O (i.e. k for α-protiated substrate in deuterium oxide/k for α-bideuterated substrate in D2O); D2Ok, solvent KIE using α-protiated substrate (i.e. k for α-protiated substrate in water/k for α-protiated substrate in D2O); D2Ok(D), solvent KIE using α-bideuterated substrate (i.e. k for α-bideuterated substrate in water/k for α-bideuterated substrate in D2O);D,D2Ok, simultaneous substrate and solvent KIE (i.e. k for α-protiated substrate in water/k for α-bideuterated substrate in D2O). The experimental kinetic parameters for Vmax and Km used to calculate the KIE are reported in supplemental Table S1.
| p-Anisyl alcohol | 2,4-Hexadien-1-ol | |
|---|---|---|
| Substrate KIE | ||
| DVmax | 5.41 ± 0.14 | 5.59 ± 0.14 |
| D(Vmax/Km) | 3.71 ± 0.25 | 9.16 ± 1.08 |
| DVmax(D2O) | 5.22 ± 0.34 | 5.57 ± 0.10 |
| D(Vmax/Km)(D2O) | 5.32 ± 0.65 | 12.10 ± 0.80 |
| Solvent KIE | ||
| D2OVmax | 1.35 ± 0.06 | 1.25 ± 0.03 |
| D2O(Vmax/Km) | 1.15 ± 0.11 | 1.26 ± 0.11 |
| D2OVmax(D) | 1.30 ± 0.07 | 1.25 ± 0.02 |
| D2O(Vmax/Km)(D) | 1.65 ± 0.17 | 1.66 ± 0.17 |
| Multiple KIE | ||
| D,D2OVmax | 7.03 ± 0.38 | 6.98 ± 0.15 |
| D,D2O(Vmax/Km) | 6.12 ± 0.58 | 15.20 ± 1.10 |