The transient and steady-state kinetic parameters for the ATPase cycle of the complex of skeletal muscle actin with smooth or skeletal muscle subfragment 1 (S-1) were determined over a wide range of actin concentrations from measurements of protein tryptophan fluorescence, the transient hydrolysis step, and the steady-state rate. The properties of the completely associated system were determined by using S-1 covalently cross-linking to actin. A four-state model was found to provide a good approximation to the kinetic and steady-state behavior. The formation of AM1T and M1T occurs by a rapid equilibrium binding of T followed by a very fast step. Actin is in rapid equilibrium with M1T and M2·DP(i), with association constants K2 and K4. The three rate constants k1, k-1, and k5 were obtained by fitting observed rate constants from transient state measurements and V(M) to the model using values of k3 and k-3 determined for S-1 alone. To fit the data for skeletal or smooth muscle acto-S-1, the calculated rate constant of the hydrolysis step k1 and the equilibrium constant K1 had to be 2-3 times smaller then the corresponding parameters (k3, K3) for S-1. The calculated effective rate constant for product release must be large for striated muscle (85 s-1 at 20°C, low ionic strength) and relatively small for smooth muscle (3 s-1). The difference in the actin-concentration dependence of association and of steady-state ATPase activity was predicted correctly from the rate constants fitted to the transient evidence. While the proposed mechanism does not exclude the possibility of additional ATP or product intermediate states, the properties of such states cannot be deduced from the kinetic evidence.
|Original language||English (US)|
|Number of pages||12|
|Journal||Journal of Biological Chemistry|
|State||Published - Jan 1 1984|
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