Steady-state ATPase activity. a Dependence of the basal steady-state ATPase activity of the myosin-7a motor on [ATP]. A hyperbolic fit to the data set (ATPase activity = (k_basal[ATP])/(K_app,actin +[ATP])) gives a value for maximal ATPase turnover (k_basal) as 0.05 ± 0.01 s⁻¹. Inset, close-up view of the data in the concentration range between 0 and 500 μM ATP. b Dependence of the steady-state ATPase activity of the human myosin-7a motor domain as a function of [F-actin]. The solid curve is a hyperbolic fit (ATPase activity = (k_cat[F-actin])/(K_app,actin + [F-actin])) giving k_cat and K_{app, actin} values of 0.35 ± 0.03 s⁻¹ and 8.3 ± 2.6 μM, respectively. The displayed data for the actin-activated steady-state ATPase activity are corrected for the basal ATPase activity. Error bars in panels A and B represent standard deviations from at least 6 determinations of each data point. The experimental conditions were as follows: 25 mM HEPES (pH 7.4), 5 mM MgCl₂, 0.5 mM DTT, and 2 mM ATP in the presence of an ATP regeneration system at a temperature of 25°C

Interaction of the myosin-7a motor with F-actin in the absence of nucleotide and in the presence of ADP. a Binding of the human myosin-7a motor domain to pyrene-actin filaments in the absence (filled circle) and presence (open circle) of saturating ADP (250 μM). The concentration of the M7a construct was kept at 1:5 to 1:7.5 of the pyrene-actin concentration. The rate of pyrene-actin quenching is linearly dependent on [pyrene-actin] up to 1.75 μM. The apparent second order rate constants derived from the slopes of linear fits to the data sets are k_+A = 3.17 ± 0.06 μM⁻¹s⁻¹ and k_+DA = 1.34 ± 0.03 μM⁻¹s⁻¹, respectively. Inset, time course upon mixing M7a (0.125 μM) with an excess of pyrene-actin (0.75 μM) approximated with a single exponential equation. Time axes are identical for the data fits and the corresponding residual plot. Error bars represent standard deviations from at least three determinations of each data point. b Chase experiment of the ternary pyrene-acto•M7a•ADP complex (0.25 μM) with excess of unlabeled F-actin (20 μM). A single exponential fit to the transient shown gives a value for k_−DA as 0.003 ± 1.10⁻⁴ s⁻¹. Time axes in the data fit and the corresponding residual plot (inset) are the same. Experimental conditions were as follows: 20°C, 25 mM MOPS (pH 7.0), 100 mM KCl, 5 mM MgCl₂, and 10 mM DTT. Nucleotide-free samples were prepared by preincubation of the reactants with 0.1 U/ml apyrase for 15 min at room temperature

ATP binding to myosin-7a motor and ATP-induced dissociation of actomyosin-7a. a ATP binding to myosin-7a motor was followed in a stopped-flow apparatus by the change in intrinsic fluorescence signal. A plot of the observed rate constants versus [ATP] shows a hyperbolic dependence converging at a rate of k₃ + k₋₃ = 23.1 ± 0.74 s⁻¹. Inset, ATP binding to myosin-7a motor monitored by tryptophan and mantATP fluorescence. Rate of ATP (filled circle) and mantATP (open circle) binding to myosin-7a motor domain as a function of nucleotide concentration. The solid lines correspond to the linear fit of the initial slopes and define the second order rate constant for ATP (K₁k₊₂ = 0.85 ± 0.02 μM⁻¹ s^_1) and mantATP (K₁k₊₂ = 1.15 ± 0.01 μM⁻¹ s^_1) binding to the myosin-7a motor. b ATP-induced dissociation of the preformed actomyosin-7a complex. Light-scattering transients were obtained by mixing actomyosin-7a with varying [ATP] under pseudo-first order conditions. A hyperbolic fit to the data set converges k₊₂ = 186 ± 5.7 s⁻¹ with half-saturating ATP concentration 1/K₁ = 681.19 ± 5.63 μM. Inset, ATP (filled circle) and mantATP (open circle) binding to actomyosin-7a. The apparent second order binding rate constants (K₁k₊₂) were deduced from the slopes of the linear approximations and are listed in Table 2. Error bars in panels a and b represent standard deviations from at least three determinations of each data point

ADP interaction. a mantADP binding to the myosin-7a motor domain. A linear approximation of the data set gives the second order rate constant k_+D = 0.98 ± 0.05 μM⁻¹ s⁻¹. Inset, time course of mantADP (1 μM) binding to M7a (0.125 μM) in a stopped-flow apparatus. A single exponential fit to the time course gives k_obs = 1.37 s⁻¹. The corresponding residual plot has the same time axes as the fit. b ADP release from M7a. Time course of the change in fluorescence intensity for the displacement of ADP (500 μM) from 0.25 μM with 1,500 μM ATP in the stopped-flow apparatus. The change in intrinsic tryptophan fluorescence was fitted using a single exponential function, giving a value of 0.3 ± 0.002 s⁻¹ for k_−D. The residual plot (inset) and the fit have the same time axes. c Competitive binding of ATP and ADP to M7a. The time course in the absence of ADP was fitted by a single exponential function, whereas the time courses in the presence of ADP were fit to bi-exponentials. The dependence of the observed amplitudes of the slow phase increases with increasing [ADP]. A hyperbolic fit of the data set resulted in a K_D of 0.52 ± 0.09 μM. Error bars represent standard deviations from at least three determinations of each data point

ADP interaction of acto•M7a. a ADP binding to acto•M7a. Linear approximation of the data set gives the second order rate constant of mantADP binding to actomyosin-7a k_+AD = 1.78 ± 0.04 μM⁻¹ s⁻¹. The corresponding intercept defines k_-AD = 0.59 ± 0.03 s⁻¹. Error bars represent standard deviations from at least three determinations of each data point. b ADP dissociation from acto•M7a. Displacement of ADP (1 μM) from 0.25 μM acto•M7a with excess ATP (1,000 μM). Single exponential approximation of the decrease in light scattering signal gives k_−AD = 0.48 ± 0.01 s⁻¹. The corresponding residual plot (inset) comprises the same time axes as the data fit

Mg²⁺ sensitivity of acto•M7a. (A) Inhibition of the rate of ADP dissociation from acto•M7a by free Mg²⁺. Increasing the concentration of free Mg²⁺ ions within the range between 0.01 and 19 mM suppresses ADP release kinetics fivefold with a K_i = 0.29 ± 0.03 mM. The data were fitted to the following equation k_−AD = (k_min·(Mg²⁺/K_i) + k_max)/((Mg²⁺/K_i) + 1). b Inhibitory effect of free Mg²⁺ ions on the actin-activated steady-state ATPase activity at 40 μM F-actin. The data set was fitted to a hyperbolic function resulting in a K_i of 0.29 mM. Error bars represent standard deviations from at least three determinations of each data point