Abstract
The rate-limiting step of the myosin basal ATPase (i.e. in absence of actin) is assumed to be a post-hydrolysis swinging of the lever arm (reverse recovery step), that limits the subsequent rapid product release steps. However, direct experimental evidence for this assignment is lacking. To investigate the binding and the release of ADP and phosphate independently from the lever arm motion, two single tryptophan-containing motor domains of Dictyostelium myosin II were used. The single tryptophans of the W129+ and W501+ constructs are located at the entrance of the nucleotide binding pocket and near the lever arm, respectively. Kinetic experiments show that the rate-limiting step in the basal ATPase cycle is indeed the reverse recovery step, which is a slow equilibrium step (k(forward) = 0.05 s(-1), k(reverse) = 0.15 s(-1)) that precedes the phosphate release step. Actin directly activates the reverse recovery step, which becomes practically irreversible in the actin-bound form, triggering the power stroke. Even at low actin concentrations the power stroke occurs in the actin-attached states despite the low actin affinity of myosin in the pre-power stroke conformation.
Highlights
The mechanism of the phosphate (Pi) release of the myosin ATPase is of particular interest, because it is the key step that is presumed to be activated by actin and is coupled to the working stroke of the cross-bridge cycle [1, 2]
This idea was supported by the experiments of Mannherz et al [5] who demonstrated that a small amount of labeled M*1⁄7ATP3 could be synthesized by the addition of millimolar concentrations of 32P-labeled phosphate to myosin subfragment 1 (S1) in the presence of saturating ADP
Phosphate Binding of W501ϩ1⁄7ADP Complex—To study ligand binding and the possible reverse reaction of the recovery step (M†1⁄7ADP1⁄7Pi 7 M*1⁄7ADP1⁄7Pi), W501ϩ was preincubated with ADP and rapidly mixed with different concentrations of a phosphate solution in the stopped-flow apparatus
Summary
M*, enhanced fluorescent state of myosin S1 (ϳ5%); M**, enhanced fluorescent state of myosin S1 (ϳ15%); S1, subfragment 1; dmATP, 3Ј-(N-methylanthraniloyl)-2Ј-deoxy-ATP; dmADP, 3Ј-(N-methylanthraniloyl)-2Ј-deoxy-ADP; Dd, D. discoideum; mant, N-methylanthraniloyl. We have made a Dd motor domain construct, called W501ϩ, which contains only a single tryptophan residue at this conserved position (Fig. 1) and gives an enhanced signal relative to the background compared with the wild-type protein (18 –20) In these studies the W501ϩ construct allowed us to separate the hydrolysis step from the preceding lever arm swing, as is described by Scheme 2. The second case would correspond to a “Brownian ratchet” mechanism, in which the pre-power stroke lever arm is freely reorienting and product release stabilizes the post-power stroke orientation We address these questions by characterizing the kinetics of ADP and phosphate binding reactions and the related lever arm swing. The low initial affinity of M*1⁄7ADP1⁄7Pi for actin increased during the lever arm swing and the subsequent product release steps, depleting the weakly bound A1⁄7M*1⁄7ADP1⁄7Pi population and pulling the reaction toward the actin-bound power stroke
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