Table 1. Kinetic constants for A3A acting on modified cytosine substrates.
| Substrate | Substituent | Size* (Å3) | k cat † (min−1) | K M (μM) | k cat/KM (μM−1min−1) | Relative activity†† (norm to –H sub) |
|---|---|---|---|---|---|---|
| C | -H | 10.5 | 41 ± 7 | 0.52 ± 0.19 | 79 | 1.0 |
| flC | -F | 16.8 | 4.9 ± 0.3 | 0.31 ± 0.05 | 16 | 0.12 ± 0.02 |
| mC | -CH3 | 30.5 | 10 ± 1 | 0.41 ± 0.13 | 25 | 0.25 ± 0.05 |
| brC | -Br | 33.0 | 0.33 ± 0.03 | 0.55 ± 0.11 | 0.060 | 8.0 (±1.5) × 10−3 |
| fC | -CHO | 36.3 | 0.011 ± 0.001 | n.c. | 2.7 (±0.5) × 10−4 | |
| iC | -I | 37.5 | 0.015 ± 0.001 | n.c. | 3.7 (±0.7) × 10−4 | |
| hmC | -CH2OH | 40.8 | 0.0073 ± 0.0004 | n.c. | 1.8 (±0.3) × 10−4 | |
| caC | -COO- | 43.1 | <0.002 | n.c. | < 4.9 (±0.8) × 10−5 |
Substrates differ only in the identity of the substituent at the 5-position of the target cytosine and are listed in order of increasing size.
* The size of each substituent (in Å3) was calculated using SPARTAN molecular modeling software.
† kcat and KM values were determined for the smallest four substrates (C, flC, mC, and brC) by performing steady-state kinetic measurements. kcat values were approximated for the four largest substrates (fC, iC, hmC, and caC) by determining the observed rate at saturating substrate concentrations. As no activity was detected on caC, the value reported corresponds to the calculated detection limit (determined in Supplementary Figure S2C). n.c., not calculated. Errors are reported as standard deviations from three independent kinetic replicates.
†† Relative activities are calculated as the ratio of kcat values, with associated error calculated from propagation of error in kcat values.