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.

Abstract

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 2 mM K-PEP, 1 mM MgCl2 and 1 mM MgCl2 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 first- order rate constant for recombination of the HMM-P-ADP complex, HMMPADP, or the S-l P-ADP complex, S-1PADP, 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, Δvo, was proportional to the value of (l-α). On the other hand, the apparent first-order rate constant for recombination of HMMPADP or S-1PADP with F-actin, vrecomb, was found to be 1/5–1/13 of Δvo. 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-MPADP 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 HMMPADP 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, Δvo was not proportional to the extent of binding of HMM with F-actin, 1-α, and Δvo was given by the equation Δ=(1-α)Δvo+αvrecomb;, where ΔVo is the value at sufficiently high concentrations of F-actin. The rate constant of the acto-HMM Mn2+ 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-MPADP without dissociation of actomyosin and the othervia 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 MPADP with F-actin. The F-actin-concentration dependence of the apparent first-order rate constant for recombination of HMMPADP with F-actin could be accounted for by the Michaelis-Menten equation. Therefore, HMMPADP 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.