Table 2.
Kinetics of target base flipping and covalent bonding upon M.HhaI interaction with cognate DNA
| Reaction | HhaI >< DNA |
HhaI + AdoHcy >< DNA + AdoHcy |
HhaI + DNA >< AdoHcy |
HhaI + AdoMet >< DNA + AdoMet |
||||
|---|---|---|---|---|---|---|---|---|
| Parameters | Rate, s−1 | Amplitude, mAU | Rate, s−1 | Amplitude, mAU | Rate, s−1 | Amplitude, mAU | Rate, s−1 | Amplitude, mAU |
| Flip-out | 27.4 ± 0.3 | 8.3 ± 0.04 | 19.1 ± 0.1 | 28 ± 0.1 | 19.7 ± 0.9 | 7.6 ± 0.5 | 120 ± 3 | 7.1 ± 0.1 |
| – | – | 2.5 ± 0.2 | 2.5 ± 0.1 | 5.5 ± 0.4 | 7.1 ± 0.4 | 14.4 ± 0.2 | 8.5 ± 0.1 | |
| C6-bond | 0.23 ± 0.06 | −0.7 ± 0.07 | 0.71 ± 0.003 | −23 ± 0.1 | 0.60 ± 0.01 | −16 ± 0.1 | 0.26 ± 0.003 | −12.8 ± 0.4 |
| Flip-back | – | – | – | – | – | – | 0.16 ± 0.004 | 5.6 ± 0.4 |
Apparent kinetic parameters derived from fitting absorption traces to multi-exponential functions for reactions involving 3 µM M.HhaI, 2.5 µM GCGC/GMGC duplex and 100 µM cofactor as shown in Figures 2 and 5. Premixed components in two chambers (separated by ><) were rapidly mixed in a stopped-flow cell to observe reaction-associated changes in absorbance. Values are reported with standard errors of the fit.