Table 1.
Reaction kinetics and product formation during BZDO single turnover.a
| Steady State |
Single Turnover | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Substrate | O2 uptake | Product formation | Rieske re-oxidation (464 nm) | ||||||||
| substrate coupled product forming oxidation |
substrate coupled nonproduct forming oxidation |
Slow oxidation phasesb | |||||||||
| pKa | kcat (s−1) | kobs (s−1) | Fractional Product Yield (%) |
RRT-1 (s−1) | % Amp | RRT-2 (s−1) | % Amp |
RRT-3 (s−1) | % Amp | % Amp | |
| benzoate | 4.2 | 4.4 ± 0.50 | 190 ± 50 | 47 ± 3 | 184 ± 23 | 49 ± 5 | 28 ± 3 | 32 ± 4 | 6.8 ± 1.0 | 14 ± 2 | 6 ± 2 |
| 4-fluoro-benzoate (4-FB) | 4.14 | 0.48 ± 0.06 | 5.2 ± 1.2 | 62 ± 5 | 5.3 ± 0.2 | 70 ± 3 | 1.3 ± 0.3 | 19 ± 2 | 11 ± 2 | ||
| 3,5-difluoro-benzoate (3,5-FB) | 3.5 | 1.0 ± 0.09 | 14.6 ± 0.6 | 63 ± 7 | 13 ± 1 | 66 ± 5 | 3.8 ± 0.6 | 22 ± 4 | 12 ± 2 | ||
| 3,4,5-trifluoro-benzoate (3,4,5-FB) | 3.46 | 0.079 ± 0.01 | 0.70 ± 0.12 | 51 ± 8 | 0.86 ± 0.08 | 66 ± 4 | 0.28 ± 0.04 | 24 ± 3 | 10 ± 2 | ||
a
Reduced BZDO was reacted with O2 saturated buffer and the substrates shown. The steady state kcat, product formation rate constant kobs, fractional yield of cis-diol product, and RRTs and amplitudes from a multiexponential fit of the time course monitored at 464 nm were determined under the experimental conditions described in Experimental Procedures.
b
Two low-amplitude phases much slower than the kcat were observed in most cases. The slowest phase has the same RRT as the substrate free reaction (0.1 s−1).
A slightly faster phase is observed only when a substrate is present, but exhibits no substrate concentration dependence.