Table 1.
Kinetic parameters of BLMHM translocation along ssDNA
| Method of determination | Value | |
|---|---|---|
| ATP hydrolysis cycle | ||
| k1 (µM−1 s−1) | mdATP | 6.9 ± 0.7 |
| k−1 (−−1) | mdATP | 120 ± 10 |
| k2 (s−1) | MDCC-PBP | >100 |
| k3 (s−1) | MDCC-PBP | >100 |
| k4 (s−1) | mdADP chasing | 5.3 ± 0.6 |
| k−4 (s−1) | mdADP chasing | ≈2 |
| k5 (s−1) | mdADP chasing | 270 ± 30 |
| mdADP binding | 110 ± 30 | |
| k−5 (µM−1 s−1) | mdADP binding | 10 ± 1 |
| ssDNA translocation | ||
| ktrans (steady-state ATP hydrolysis rate constant during translocation, s−1) | PK/LDH assay, modeling | 33 ± 2 |
| MDCC-PBP | 27 ± 2 | |
| kend (steady-state ATP hydrolysis rate constant at 5′-end, s−1) | PK/LDH assay, modeling | 5.6 ± 0.5 |
| koff, int (dissociation from ssDNA during translocation, s−1) | Trp fluorescence | ≈0.2 |
| koff, end (dissociation from 5′-end, s−1) | Trp fluorescence, PK/LDH assay, modeling | 2.7 ± 0.3 |
| P (processivity: probability of taking the next translocation step)a | Calculated | 0.98 |
| b (binding site size in nucleotides) | HEX fluorescence, mdADP, PK/LDH assay | 14 |
| MDCC-PBP | 12.4 ± 0.1 | |
| s (translocation step size in nucleotides) | MDCC-PBP | 1.1 ± 0.1 |
| ATP coupling ratio (ATP consumed/nucleotide traveled) | MDCC-PBP | 0.87 ± 0.08 |
aThe mean number of translocation steps taken in a single run is P/(1 − P) = 50.
Nomenclature refers to Figure 1. Means ± SEM values are shown for n = 3.