Table 5.
Effect of phosphate neutralization on the EcoRV-DNA transition state complex.
| Protein | DNAa | k1 (s−1)b | kref/k1 | ΔΔG1°‡ (kcal/mol)c | k2 (s−1)b | kref/k2 | ΔΔG2°‡ (kcal/mol)c |
|---|---|---|---|---|---|---|---|
| wild type | GATATC | 1.8 ± 0.1 | 1 | 0 | 1.8 ± 0.1 | 1 | 0 |
| wild type | PMe-5 | 1.1 ± 0.1 | 2 | +0.3 ± 0.1 | 1.2 ± 0.1 | 2 | +0.3 ± 0.1 |
| wild type | PMe-4 | 0.26 ± 0.04 | 7 | +1.1 ± 0.1 | 0.26 ± 0.04 | 7 | +1.1 ± 0.1 |
| wild type | PMe-3 | 1.3 ± 0.2 | 1 | +0.2 ± 0.1 | 1.3 ± 0.2 | 1 | +0.2 ± 0.1 |
| wild type | PMe-2 | 0.05 ± 0.004 | 36 | +2.1 ± 0.1 | 0.05 ± 0.008 | 36 | +2.1 ± 0.1 |
| wild type | Rp-PMe-2 | 0.08 ± 0.01 | 22 | +1.8 ± 0.1 | 0.09 ± 0.01 | 22 | +1.7 ± 0.1 |
| CONCAVE FACE | |||||||
| R226A | GATATC | 0.06 ± 0.01 | 30 | +2.0 ± 0.1 | 0.06 ± 0.01 | 30 | +2.0 ± 0.1 |
| R226A | PMe-5 | 0.28 ± 0.05 | 6 | +1.1 ± 0.2 | 0.33 ± 0.02 | 6 | +1.1 ± 0.2 |
| K119A | GATATC | 0.13 ± 0.01 | 14 | +1.6 ± 0.1 | 0.14 ± 0.01 | 14 | +1.5 ± 0.1 |
| K119A | Rp-PMe-2 | 0.56 ± 0.03 | 3 | +0.7 ± 0.1 | 0.61 ± 0.05 | 3 | +0.6 ± 0.1 |
| R221A | GATATC | 1.4 ± 0.8 | 0.8 | +0.2 ± 0.2 | 1.3 ± 0.8 | 1 | +0.2 ± 0.2 |
| R221A | Sp-PMe-2 | 0.006 ± 0.001 | 300 | +3.4 ± 0.1 | 0.006 ± 0.001 | 300 | +3.4 ± 0.1 |
| CONVEX FACE | |||||||
| R140A | GATATC | 0.37 ± 0.1 | 5 | +0.9 ± 0.2 | 0.32 ± 0.1 | 5 | +1.0 ± 0.2 |
| R140A | PMe+3 | 0.02 ± 0.001 | 90 | +2.7 ± 0.1 | 0.02 ± 0.002 | 90 | +2.6 ± 0.1 |
a
First order cleavage rate constants were measured for the double-stranded oligonucleotide d(TGTGTTGTAGGATATCCTACAGGT) in binding buffer + 0.1 M K+. Reactions at 295K were initiated by the addition of 10 mM MgCl2.
b
k1 denotes the first order cleavage rate constant for the first (top) strand and k2 that for the second (bottom) strand..
c
ΔΔGn°‡ = −RTln(kn/kref)