Mechanism of the Mg²⁺- and Mn²⁺-ATPase Reactions of Acto-H-Meromyosin and Acto-Subfragment-1 in the Absence of KCl at Room Temperature: Direct Decomposition of the Complex of Myosin-P-ADP with F-Actin

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The extent of binding of myosin heads with F-actin was estimated by a light-scattering and an ultracentrifugal separation method, using well-homogenized F-actin. The extent of binding of HMM or S-1 with F-actin during the ATPase reaction was estimated in 2mM K-PEP, 1 mM MgCl₂, and 1mM K-P₁ at pH 7.4 and compared with the rate of the acto-HMM ATPase or acto-S-1 ATPase [EC 3. 6. 1. 3] reaction in the steady state. The apparent firstorder rate constant for recombination of the HMM-P-ADP complex, HMM^ADP_P, or the S-1-P-ADP complex, S-1^ADPP with F-actin was also determined.
At 20°C, the extent of binding (1-α) increased with increase in the F-actin concentration and approached 1.0 at a sufficiently high concentration of F-actin. The steady-state rate constant of the F-actin-dependent ATPase reaction, Δv₀, was proportional to the value of (1-α). On the other hand, the apparent first-order rate constant for recombination of HMM^ADP_P or S-1^ADP_P with F-actin, v_recomb, was found to be 1/5-1/13 of Δv₀. Therefore, we concluded that the main route of ATP hydrolysis at low ionic strength at 20°C is the one via direct decomposition of acto-M^ADP_P and that the ATP hydrolysis cycle does not involve the dissociation step of actomyosin. This conclusion was supported by our finding that a high ATPase activity was observed immediately after adding ATP to acto-HMM, while a high ATPase rate in the steady state was observed after a lag phase required for binding of HMM^ADP_P with F-actin, when the acto-HMM ATPase reaction was started by adding F-actin to a solution containing HMM and ATP.
At 12°C, the rate constant of the acto-HMM ATPase reaction, Δv₀, was not proportional to the extent of binding of HMM with F-actin, 1-α, and Δv₀ was given by the equation Δv₀=(1-α)ΔV₀+αv_recomb, where ΔV₀. is the value at sufficiently high concentrations of F-actin. The rate constant of the acto-HMM Mn²⁺-ATPase reaction at low ionic strength at 25°C was also accounted for by the above equation.
The results obtained in this study, together with those described in one of the preceding papers (Ikebe, M., Inoue, A., & Tonomura, Y. (1980) J. Biochem. 88, 1653-1662), clearly demonstrate that in the actomyosin ATPase reaction, ATP is hydrolyzed via two routes: one via direct decomposition of acto-M^ADP_P without dissociation of actomyosin and the other via the route proposed by Lymn and Taylor (Lymn, R. W. & Taylor, E. W. (1971) Biochemistry 10, 4617-4624). The rate of the latter route is limited by the steps for recombination of M^ADP_P with F-actin.
The F-actin-concentration dependence of the apparent first-order rate constant for recombination of HMM^ADP_P with F-actin could be accounted for by the Michaelis-Menten equation. Therefore, HMM^ADP_P produced by the reaction of HMM with ATP may exist largely in a refractory state and may recombine with F-actin only after transformation into a nonrefractory state.