Table 4.
Comparison of mechanics and biochemistry: potential rate-limiting steps
| Fibre shortening and cross-bridge dissociation | ||||
|---|---|---|---|---|
| Substrate | Velocity (L s−1)a | NDP koff (s−1)b | Predicted velocity (L s−1)c | k′ (AM) (μm−1 s−1) |
| Mg·ATP | 0.88 ± 0.04 (19) | 300 ± 50 (4) | 1.8 | 1.2 ± 0.1 (6) |
| Mn·ATP | 0.73 ± 0.04 (6) | 300 ± 50 (4) | 1.8 | 1.2 ± 0.2 (4) |
| Ni·ATP | 0.17 ± 0.02 (8) | 150 ± 40 (4) | 0.9 | 0.2 ± 0.05 (4) |
| Mg·CTP | 0.51 ± 0.04 (5) | > 300d | > 1.8 | 0.14, 0.07e |
| Mg·ITP | 0.13 (2) | ? | ? | ? |
| Force recovery, ATP hydrolysis and phosphate release | ||||||
|---|---|---|---|---|---|---|
| Substrate | kr1 (s−1)a | k+hyd (s−1) | Khyd | kPi-off (s−1) | Predicted kr1f (s−1) | NTPase (s−1) |
| Mg·ATP | 5.2 ± 0.3 (46) | 7.7 ± 0.9 (8) | 1.7 (8) | 23h | 5.2 | 1.9 ± 0.2 (11) |
| Mn·ATP | 4.6 ± 0.2 (14) | 8.8 ± 0.9 (8) | 5.8 (8) | ? | ? | 2.5 ± 0.2 (8) |
| Ni·ATP | 3.2 ± 0.2 (15) | > 0.6g (8) | > 0.006g (8) | ? | ? | 0.7 ± 0.2 (4) |
| Mg·CTP | 7.8 ± 0.4 (11) | 15.4h | 3.3h | 18h | 7.8 | 2.2 ± 0.1 (5) |
| Mg·ITP | 1.0 ± 0.1 (2) | 0.15i | ∼0.5i | ? | ? | 0.15 ± 0.04 (5) |
Values are mean ± error, where error is s.e.m. for mechanics and s.d. for biochemistry. Number of replicates (n) shown in parentheses (= fibres in mechanics experiments). NDP koff, first-order rate constant of ADP dissociation from actomyosin (n indicates replicates at each [ATP]); k′, second-order rate constant of Me·NTP binding to actomyosin; kr1, single-exponential rate constant of force recovery; k+hyd and Khyd, forward rate and equilibrium constants, respectively, of the NTP hydrolysis step of myosin S1. NTPase, steady-state NTPase activity of cross-linked acto-S1.
For Mg·ATP, shortening velocity and kr1 are taken from Table 2. The mean ± s.e.m. for the alternative substrates was obtained by multiplying the Mg·ATP values by the relative effects given in Table 3. L s−1, fibre lengths per second.
Equilibrium constant for ADP binding to actomyosin = 0.13 mm for Mg·ATP. n number of traces at each [ATP].
Lengths per second calculated assuming 5 nm working stroke estimated from shortening velocity at low [ATP] (White et al. 1993).
Rate of CDP release reported by Robinson et al. (1993) was higher than for ADP release (taken from this study).
White et al. (1993) using actomyosin-S1 and Regnier et al. (1998) using acto-heavy meromyosin (acto-HMM), respectively; 10°C.
Predicted kr1 is from single-exponential functions fitted to simulated force records obtained from the kinetic scheme presented in Discussion, where hydrolysis is step 2 and Pi release is step 4. For Mg·ATP, the rate constants are given there and above, with K3= 0.5. For Mg·CTP, K3= 1, and in addition to the rates given above, k1= 240 s−1(White et al. 1993) and k5= 0.7 s−1 (taken from fibre CTPase; Pate et al. 1993). k1 and k5 have been corrected for temperature.
k+hyd for Ni·ATP is a lower estimate as the maximum rate was not reached within the accessible [S1].
White et al. (1997). Khyd is that reported for S1 at 10°C; k+hyd has been extrapolated from 10 to 5°C using a Q10 of 5 and correcting for ionic strength. The Pi release rate for CTP assumes that the ratio of rates using CTP and ATP is the same at 20°C (their study) and 5°C.
k+hyd for ITP was estimated from Khyd and steady-state ITPase activity at 20°C, where the hydrolysis step is rate limiting. k+hyd and Khyd have been corrected for temperature and ionic strength.