Table 1. Functional properties of myosin 5a constructsa.
| Parameter | Method of determination | wt | I67K |
| Steady-state ATPase activity | NADH assay | ||
| Basal activity (s−1)b | 0.020±0.03 | 0.015±0.04 | |
| Actin-activated k cat (s−1) | 9.9±0.8 | 1.8±0.4 | |
| K actin (µM)c | 5.2±0.3 | 1.0±0.4 | |
| K ATP (µM)d | 12±1 | 2.5±0.3 | |
| Motility | |||
| Actin gliding (nm•s−1)e | Actin gliding assay | 410±50 | 82±22 |
| v max (nm•s−1) | TIRF assay | 410±30 | 250±40 |
| K ATP (µM) | TIRF assay | 11±0.4 | 130±10 |
| Trp fluorescence change on nucleotide binding (%) | Trp fluorescence | ||
| ATP | 17±3 | 9±1 | |
| AMPPNP | 12±1 | 13±3 | |
| ATPγS | 0±1 | –1±1 | |
| ADP | –1±1 | 1±2 | |
| ATP binding and hydrolysis | |||
| K 1 k 2 (µM−1s−1)f | Trp fluorescence | 1.3±0.2 | 1.3±0.2 |
| k 3+ k –3 (s−1)f | Trp fluorescence | 270±60 | 760±50 |
| k 3+ k –3 (s−1)g | Quenched-flow | >35 | >39 |
| K 3 | Quenched-flow | 0.64±0.05 | 1.8±0.6 |
| K 1’k 2’ (µM−1s−1)f | PA fluorescence, light scattering | 0.73±0.08 | 1.2±0.2 |
| k 2’ (s−1)f | PA fluorescence, light scattering | >200 | >300 |
| Pi release | MDCC-PBP fluorescence | ||
| k 4 (s−1)h | 0.029±0.003 | 0.025±0.002 | |
| k 4’ (s−1)i | ≥32 | ≥12 | |
| A max (mol Pi/mol m5aS1)j | 0.99±0.01 | 0.90±0.02 | |
| ADP interaction | |||
| k 5 (s−1) | mdADP fluorescence | 2.4±0.1 | 4.3±0.6; 1.3±0.1k |
| k 5’ (s−1) | PA fluorescence | 17±3 | 2.7±0.1 |
| K 5’ (µM) | PA fluorescence | 3.7±0.8 | 1.0±0.3 |
| Actin interaction | PA fluorescence | ||
| k –6 (µM−1s−1) | 28±1l | 7.3±0.2 | |
| k 6 (s−1) | 1.1×10−3l | (8.1±0.8)×10−3 | |
| K 6 (µM) | 3.9×10−5l | 1.1×10−3 |
Solution kinetic data are for m5aS1 and motility data are for m5aHMM. Nomenclature of kinetic constants refers to Fig. 1B . All parameters were measured at 25°C. Mean ± SD values for two to four independent experiments are shown.
At 1 mM ATP.
Half-saturating actin concentration (at 1 mM ATP).
Half-saturating ATP concentration (at 10 µM actin).
At 1 mM ATP.
Results of exponential analysis are shown ( Fig. 2 ; rapid phase data for I67K-m5aS1). See also Fig. S1 for global fitting results on I67K-m5aS1.
Lower bounds set by maximal rate constant of rapid pre-steady state burst at 30 µM ATP.
From steady-state slopes recorded at 100 µM ATP.
Lower bounds set by maximal rate constant of rapid pre-steady state burst at 10 µM actin.
Maximal amplitude of rapid pre-steady state burst at 10 µM actin.
Rate constants of the rapid (40±3% amplitude) and slow phases of biphasic mdADP release.
From ref. [17].