Chemical Decoupling of ATPase Activation and Force Production from the Contractile Cycle in Myosin by Steric Hindrance of Lever-Arm Movement

The myosin motor protein generates force in muscle by hydrolyzing Adenosine 5′-triphosphate (ATP) while interacting transiently with actin. Structural evidence suggests the myosin globular head (subfragment 1 or S1) is articulated with semi-rigid catalytic and lever-arm domains joined by a flexible converter domain. According to the prevailing hypothesis for energy transduction, ATP binding and hydrolysis in the catalytic domain drives the relative movement of the lever arm. Actin binding and reversal of the lever-arm movement (power stroke) applies force to actin. These domains interface at the reactive lysine, Lys84, where trinitrophenylation (TNP-Lys84-S1) was observed in this work to block actin activation of myosin ATPase and in vitro sliding of actin over myosin. TNP-Lys84-S1's properties and interactions with actin were examined to determine how trinitrophenylation causes these effects. Weak and strong actin binding, the rate of mantADP release from actomyosin, and actomyosin dissociation by ATP were equivalent in TNP-Lys84-S1 and native S1. Molecular dynamics calculations indicate that lever-arm movement inhibition during ATP hydrolysis and the power stroke is caused by steric clashes between TNP and the converter or lever-arm domains. Together these findings suggest that TNP uncouples actin activation of myosin ATPase and the power stroke from other steps in the contraction cycle by inhibiting the converter and lever-arm domain movements.     

Mechanochemical coupling in actomyosin energy transduction studied by in vitro movement assay

In order to study the mechanochemical coupling in actomyosin energy transduction, the sliding distance of an actin filament induced by one ATP hydrolysis cycle was obtained by using an in vitro movement assay that permitted quantitative and simultaneous measurements of (1) the movements of single fluorescently labeled actin filaments on myosin bound to coverslip surfaces and (2) the ATPase rates. The sliding distance was determined as (the working stroke time in one ATPase cycle, tws) x (the filament velocity, v). tws was obtained from the ATPase turnover rate of myosin during the sliding (kt), the ATP hydrolysis time (delta t) and the ON-rate at which myosin heads enter into the working stroke state when they encounter actin (kON); tws approximately 1/kt-delta t-1/kON. kt was estimated from the ATPase rates of the myosin-coated surface during the sliding of actin filaments. delta t has been determined as less than 1/100 per second, kON was estimated by analyzing the movements of very short (40 nm) filaments. The resulting sliding distance during one ATP hydrolysis cycle near zero load was greater than 100 nm, which is about ten times longer than that expected for a single attachment-detachment cycle between an actin and a myosin head. This leads to the conclusion that the coupling between the ATPase and attachment-detachment cycles is not determined rigidly in a one-to-one fashion.     

The myosin cardiac loop participates functionally in the actomyosin interaction

The motor protein myosin in association with actin transduces chemical free energy in ATP into work in the form of actin translation against an opposing force. Mediating the actomyosin interaction in myosin is an actin binding site distributed among several peptides on the myosin surface including surface loops contributing to affinity and actin regulation of myosin ATPase. A structured surface loop on beta-cardiac myosin, the cardiac or C-loop, was recently demonstrated to affect myosin ATPase and was indirectly implicated in the actomyosin interaction. The C-loop is a conserved feature of all myosin isoforms with crystal structures, suggesting that it is an essential part of the core energy transduction machinery. It is shown here that proteolytic digestion of the C-loop in beta-cardiac myosin eliminates actin-activated myosin ATPase and reduces actomyosin affinity in rigor more than 100-fold. Studies of C-loop function in smooth muscle myosin were also undertaken using site-directed mutagenesis. Mutagenesis of a single charged residue in the C-loop of smooth muscle myosin alters actomyosin affinity and doubles myosin in vitro motility and actin-activated ATPase velocities, thereby involving a charged region of the loop in the actomyosin interaction. It appears likely that the C-loop is an essential electrostatic binding site for actin involved in modulation of actomyosin affinity and regulation of actomyosin ATPase velocity.     

A kinetic model that explains the effect of inorganic phosphate on the mechanics and energetics of isometric contraction of fast skeletal muscle

A conventional five-step chemo-mechanical cycle of the myosin–actin ATPase reaction, which implies myosin detachment from actin upon release of hydrolysis products (ADP and phosphate, Pi) and binding of a new ATP molecule, is able to fit the [Pi] dependence of the force and number of myosin motors during isometric contraction of skeletal muscle. However, this scheme is not able to explain why the isometric ATPase rate of fast skeletal muscle is decreased by an increase in [Pi] much less than the number of motors. The question can be solved assuming the presence of a branch in the cycle: in isometric contraction, when the force generation process by the myosin motor is biased at the start of the working stroke, the motor can detach at an early stage of the ATPase cycle, with Pi still bound to its catalytic site, and then rapidly release the hydrolysis products and bind another ATP. In this way, the model predicts that in fast skeletal muscle the energetic cost of isometric contraction increases with [Pi]. The large dissociation constant of the product release in the branched pathway allows the isometric myosin–actin reaction to fit the equilibrium constant of the ATPase.     

The rate-limiting step in the actomyosin adenosinetriphosphatase cycle

We have previously shown that myosin does not have to detach from actin during each cycle of ATP hydrolysis. In the present study, using the A-1 isoenzyme of myosin subfragment 1, we have investigated the nature of the rate-limiting steps in the ATPase cycle. Our results show that, at 15 degrees C, at very low ionic strength, KATPase determined from the double-reciprocal plot of ATPase activity vs. actin concentration is more than 6-fold stronger than KBINDING determined by directly measuring the binding of A-1 myosin subfragment 1 to actin during steady-state ATP hydrolysis. Computer modeling shows that this large difference between KATPase and KBINDING is not compatible with Pi release being the rate-limiting step in the ATPase cycle. If Pi release is not rate limiting, it is possible that the ATP hydrolysis step, itself, is rate limiting. However, this predicts that, at high actin concentration, the value of the initial Pi burst should be close to zero. Therefore, we measured the magnitude of the initial Pi burst in the presence of actin, using both direct measurement and measurement of relative fluorescence magnitude. Our results suggest that the magnitude of the initial Pi burst in the presence of actin is considerably higher than would be expected if the ATP hydrolysis step were the rate-limiting step in the ATPase cycle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural rearrangements in the active site of smooth-muscle myosin

Structural rearrangements of the myosin upper-50 kD subdomain are thought to play a key role in coordinating actin binding with nucleotide hydrolysis during the myosin ATPase cycle. Such rearrangements could open and close the active site in opposition to the actin-binding cleft, helping explain the opposing affinities of myosin for actin and nucleotide. To directly examine conformational changes across the active site during the ATPase cycle we have genetically engineered a mutant of chicken smooth-muscle myosin, F344W motor domain essential light chain, which contains a single tryptophan (344W) located on a short loop between two alpha helixes that traverse the upper-50 kD subdomain in front of the active site. Fluorescence resonance energy transfer was examined between the 344W donor probe and 2'(3')-O-(N-methylanthraniloyl) (mant)-nucleotide acceptor probes in the active site of this construct. The observed fluorescence resonance energy transfer efficiencies were 6.4% in the presence of mant ADP and 23.8% in the presence of mant ATP, corresponding to distances of 33.4 A and 24.9 A, respectively. Our results are consistent with structural rearrangements in which there is an 8.5-A closure between the 344W residue and the mant moiety during the transition from the strongly (ADP) to weakly (ATP) actin-bound states of the myosin ATPase cycle.     

Two conserved lysines at the 50/20-kDa junction of myosin are necessary for triggering actin activation

Actin stimulates myosin's activity by inducing structural alterations that correlate with the transition from a weakly to a strongly bound state, during which time inorganic phosphate (P(i)) is released from myosin's active site. The surface loop at the 50/20-kDa junction of myosin (loop 2) is part of the actin interface. Here we demonstrate that elimination of two highly conserved lysines at the C-terminal end of loop 2 specifically blocks the ability of heavy meromyosin to undergo a weak to strong binding transition with actin in the presence of ATP. Removal of these lysines has no effect on strong binding in the absence of nucleotide, on the rate of ADP binding or release, or on the basal ATPase activity. We further show that the 16 amino acids of loop 2 preceding the lysine-rich region are not essential for actin activation, although they do modulate myosin's affinity for actin in the presence of ATP. We conclude that interaction of the conserved lysines with acidic residues in subdomain 1 of actin either triggers a structural change or stabilizes a conformation that is necessary for actin-activated release of P(i) and completion of the ATPase cycle.     

Analysis of the conformational change of myosin during ATP hydrolysis using fluorescence resonance energy transfer

In order to elucidate the molecular basis of energy transduction by myosin as a molecular motor, a fluorescent ribose-modified ATP analog 2'(3')-O-[6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl]-ATP (NBD-ATP), was utilized to study the conformational change of the myosin motor domain during ATP hydrolysis using the fluorescence resonance energy transfer (FRET) method. The FRET efficiency from the fluorescent probe, BD- or AD-labeled at the reactive cysteine residues, SH1 (Cys 707) or SH2 (Cys697), respectively, to the NBD fluorophore in the ATP binding site was measured for several transient intermediates in the ATPase cycle. The FRET efficiency was greater than that using NBD-ADP. The FRETs for the myosin.ADP.AlF4- and myosin.ADP.BeFn ternary complexes, which mimic the M*.ADP.P(i) state and M.ATP state in the ATPase cycle, respectively, were similar to that of NBD-ATP. This suggests that both the SH1 and SH2 regions change their localized conformations to move closer to the ATPase site in the M*.ATP state and M**.ADP.P(i) state than in the M*.ADP state. Furthermore, we measured energy transfer from BD in the essential light chain to NBD in the active site. Assuming the efficiency at different states, myosin adopts a conformation such that the light chain moves closer to the active site by approximately 9 A during the hydrolysis of ATP.     

The structural basis of myosin V processive movement as revealed by electron cryomicroscopy

The processive motor myosin V has a relatively high affinity for actin in the presence of ATP and, thus, offers the unique opportunity to visualize some of the weaker, hitherto inaccessible, actin bound states of the ATPase cycle. Here, electron cryomicroscopy together with computer-based docking of crystal structures into three-dimensional (3D) reconstructions provide the atomic models of myosin V in both weak and strong actin bound states. One structure shows that ATP binding opens the long cleft dividing the actin binding region of the motor domain, thus destroying the strong binding actomyosin interface while rearranging loop 2 as a tether. Nucleotide analogs showed a second new state in which the lever arm points upward, in a prepower-stroke configuration (lever arm up) bound to actin before phosphate release. Our findings reveal how the structural elements of myosin V work together to allow myosin V to step along actin for multiple ATPase cycles without dissociating.     

Force generation, work, and coupling in molecular motors.

A mechanism is proposed for molecular motors in which force is generated by a protein conformational change driven by binding energy (in muscle, that of myosin with actin as well as with ATP, ADP, or Pi). Work, the product of the force generated by one myosin or kinesin molecule (F) and the distance over which it acts (d), is a function of a ratio of dissociation constants before and after the contractile step: F.d < RT ln(KAe/KAc). From published data the ratio is > 2 x 10(4), which can be explained by conversion of a surface complex to an enclosed, or partly enclosed, complex. Although the complex performing the work stroke is in unstrained conformation, the complex after the work stroke is much more stable, owing to binding forces; the latter, however, is destabilized by the load, which thereby opposes the contractile conformational change, countering the force-generating reaction. The connection between the free energy release and work is implicit in the mechanism, inasmuch as coupling, like force generation, depends on conformational changes driven by binding energy (internal rather than external work being involved in coupling). The principles apply whether ATP or an ion gradient drives the system. At high load, in muscle, the mechanism allows for a summation of the forces generated by several myosin molecules.     

Energy transduction optical sensor in skeletal myosin

The skeletal myosin cross-bridge in dynamic association with actin is the unitary energy transducer in muscle, converting free energy from ATP hydrolysis into contractile force. Myosin's conserved ATP-sensitive tryptophan (AST) is an energy transduction optical sensor signaling transduction-related transient conformation change by modulating its fluorescence intensity amplitude and relaxation rate. Recently introduced techniques have provided the means of observing the time-resolved intensity decay from this single residue in the native protein to elucidate the mechanism of its ATP sensitivity. AST signal characteristics could be derived from local protein structure by a scenario involving interactions with excited-state tryptophan. This investigation suggests the very different possibility that hypochromism induced in the tryptophan absorption band, a ground-state effect, is a significant structural effector of optical transduction sensing. This possibility makes feasible the interpretation of the transient AST optical signal in terms of dynamical protein structure, thereby raising the empirical signal to the level of a structural determinant. Using the crystallographically based geometry from several myosin structures, the maximum calculated AST hypochromism is <10% to be compared with the value of approximately 30% observed here experimentally. Rationalizing the discrepancy invites further investigation of S1 dynamical structure local to the AST during transduction.     

Insights into the chemomechanical coupling of the myosin motor from simulation of its ATP hydrolysis mechanism

The molecular motor myosin converts chemical energy from ATP hydrolysis into mechanical work, thus driving a variety of essential motility processes. Although myosin function has been studied extensively, the catalytic mechanism of ATP hydrolysis and its chemomechanical coupling to the motor cycle are not completely understood. Here, the catalysis mechanism in myosin II is examined using quantum mechanical/molecular mechanical reaction path calculations. The resulting reaction pathways, found in the catalytically competent closed/closed conformation of the Switch-1/Switch-2 loops of myosin, are all associative with a pentavalent bipyramidal oxyphosphorane transition state but can vary in the activation mechanism of the attacking water molecule and in the way the hydrogens are transferred between the heavy atoms. The coordination bond between the Mg2+ metal cofactor and Ser237 in the Switch-1 loop is broken in the product state, thereby facilitating the opening of the Switch-1 loop after hydrolysis is completed, which is required for subsequent strong rebinding to actin. This reveals a key element of the chemomechanical coupling that underlies the motor cycle, namely, the modulation of actin unbinding or binding in response to the ATP or ADP x P(i) state of nucleotide-bound myosin.     

An unusual transduction pathway in human tonic smooth muscle myosin

The motor protein myosin binds actin and ATP, producing work by causing relative translation of the proteins while transducing ATP free energy. Smooth muscle myosin has one of four heavy chains encoded by the MYH11 gene that differ at the C-terminus and in the active site for ATPase due to alternate splicing. A seven-amino-acid active site insert in phasic muscle myosin is absent from the tonic isoform. Fluorescence increase in the nucleotide sensitive tryptophan (NST) accompanies nucleotide binding and hydrolysis in several myosin isoforms implying it results from a common origin within the motor. A wild-type tonic myosin (smA) construct of the enzymatic head domain (subfragment 1 or S1) has seven tryptophan residues and nucleotide-induced fluorescence enhancement like other myosins. Three smA mutants probe the molecular basis for the fluorescence enhancement. W506+ contains one tryptophan at position 506 homologous to the NST in other myosins. W506F has the native tryptophans except phenylalanine replaces W506, and W506+(Y499F) is W506+ with phenylalanine replacing Y499. W506+ lacks nucleotide-induced fluorescence enhancement probably eliminating W506 as the NST. W506F has impaired ATPase activity but retains nucleotide-induced fluorescence enhancement. Y499F replacement in W506+ partially rescues nucleotide sensitivity demonstrating the role of Y499 as an NST facilitator. The exceptional response of W506 to active site conformation opens the possibility that phasic and tonic isoforms differ in how influences from active site ATPase propagate through the protein network.     

Switch I closure simultaneously promotes strong binding to actin and ADP in smooth muscle myosin

The motor protein myosin uses energy derived from ATP hydrolysis to produce force and motion. Important conserved components (P-loop, switch I, and switch II) help propagate small conformational changes at the active site into large scale conformational changes in distal regions of the protein. Structural and biochemical studies have indicated that switch I may be directly responsible for the reciprocal opening and closing of the actin and nucleotide-binding pockets during the ATPase cycle, thereby aiding in the coordination of these important substrate-binding sites. Smooth muscle myosin has displayed the ability to simultaneously bind tightly to both actin and ADP, although it is unclear how both substrate-binding clefts could be closed if they are rigidly coupled to switch I. Here we use single tryptophan mutants of smooth muscle myosin to determine how conformational changes in switch I are correlated with structural changes in the nucleotide and actin-binding clefts in the presence of actin and ADP. Our results suggest that a closed switch I conformation in the strongly bound actomyosin-ADP complex is responsible for maintaining tight nucleotide binding despite an open nucleotide-binding pocket. This unique state is likely to be crucial for prolonged tension maintenance in smooth muscle.     

Cooperative actions between myosin heads bring effective functions

A recent study with single molecule measurements has reported that muscle myosin, a molecular motor, stochastically generates multiple steps along an actin filament associated with the hydrolysis of a single ATP molecule [Kitamura, K., Tokunaga, M., Esaki, S., Iwane, A.H., Yanagida, T., 2005. Mechanism of muscle contraction based on stochastic properties of single actomyosin motors observed in vitro. Biophysics 1, 1-19]. We have built a model reproducing such a stochastic movement of a myosin molecule incorporated with ATPase reaction cycles and demonstrated that the thermal fluctuation was a key for the function of myosin molecules [Esaki, S., Ishii, Y., Yanagida, T., 2003. Model describing the biased Brownian movement of myosin. Proc. Jpn. Acad. 79 (Ser B), 9-14]. The size of the displacement generated during the hydrolysis of single ATP molecules was limited within a half pitch of an actin filament when a single myosin molecules work separately. However, in muscle the size of the displacement has been reported to be greater than 60 nm [Yanagida, T., Arata, T., Oosawa, F., 1985. Sliding distance of actin filament induced by a myosin crossbridge during one ATP hydrolysis cycle. Nature 316, 366-369; Higuchi et al., 1991]. The difference suggests cooperative action between myosin heads in muscle. Here we extended the model built for an isolated myosin head to a system in which myosin heads are aligned in muscle arrangement to understand the cooperativity between heads. The simulation showed that the rotation of the actin filament [Takezawa, Y., Sugimoto, Y., Wakabayashi, K., 1998. Extensibility of the actin and myosin filaments in various states of skeletal muscles as studied by X-ray diffraction. Adv. Exp. Med. Biol. 453, 309-317; Wakabayashi, K., Ueno, Y., Takezawa, Y., Sugimoto, Y., 2001. Muscle contraction mechanism: use of X-ray synchrotron radiation. Nat. Enc. Life Sci. 1-11] associated with the release of ATPase products and binding of ATP as well as interaction between myosin heads allowed the myosin filament to move greater than a half pitch of the actin filament while a single ATP molecule is hydrolyzed. Our model demonstrated that the movement is loosely coupled to the ATPase cycle as observed in muscle.     

The post-rigor structure of myosin VI and implications for the recovery stroke

Myosin VI has an unexpectedly large swing of its lever arm (powerstroke) that optimizes its unique reverse direction movement. The basis for this is an unprecedented rearrangement of the subdomain to which the lever arm is attached, referred to as the converter. It is unclear at what point(s) in the myosin VI ATPase cycle rearrangements in the converter occur, and how this would effect lever arm position. We solved the structure of myosin VI with an ATP analogue (ADP.BeF3) bound in its nucleotide-binding pocket. The structure reveals that no rearrangement in the converter occur upon ATP binding. Based on previously solved myosin structures, our structure suggests that no reversal of the powerstroke occurs during detachment of myosin VI from actin. The structure also reveals novel features of the myosin VI motor that may be important in maintaining the converter conformation during detachment from actin, and other features that may promote rapid rearrangements in the structure following actin detachment that enable hydrolysis of ATP.     

Drosophila myosin VIIA is a high duty ratio motor with a unique kinetic mechanism

Mutations of myosin VIIA cause deafness in various species from human and mice to Zebrafish and Drosophila. We analyzed the kinetic mechanism of the ATPase cycle of Drosophila myosin VIIA by using a single-headed construct with the entire neck domain. The steady-state ATPase activity (0.06 s(-1)) was markedly activated by actin to yield V(max) and K(ATPase) of 1.72 s(-1) and 3.2 microm, respectively. The most intriguing finding is that the ATP hydrolysis predominantly takes place in the actin-bound form (actin-attached hydrolysis) for the actomyosin VIIA ATPase reaction. The ATP hydrolysis rate was much faster for the actin-attached form than the dissociated form, in contrast to other myosins reported so far. Both the ATP hydrolysis step and the phosphate release step were significantly faster than the entire ATPase cycle rate, thus not rate-determining. The rate of ADP dissociation from actomyosin VIIA was 1.86 s(-1), which was comparable with the overall ATPase cycle rate, thus assigned to be a rate-determining step. The results suggest that Drosophila myosin VIIA spends the majority of the ATPase cycle in an actomyosin.ADP form, a strong actin binding state. The duty ratio calculated from our kinetic model was approximately 0.9. Therefore, myosin VIIA is classified to be a high duty ratio motor. The present results suggested that myosin VIIA can be a processive motor to serve cargo trafficking in cells once it forms a dimer structure.     

An ionic–chemical–mechanical model for muscle contraction

The dynamic process underlying muscle contraction is the parallel sliding of thin actin filaments along an immobile thick myosin fiber powered by oar‐like movements of protruding myosin cross bridges (myosin heads). The free energy for functioning of the myosin nanomotor comes from the hydrolysis of ATP bound to the myosin heads. The unit step of translational movement is based on a mechanical‐chemical cycle involving ATP binding to myosin, hydrolysis of the bound ATP with ultimate release of the hydrolysis products, stress‐generating conformational changes in the myosin cross bridge, and relief of built‐up stress in the myosin power stroke. The cycle is regulated by a transition between weak and strong actin–myosin binding affinities. The dissociation of the weakly bound complex by addition of salt indicates the electrostatic basis for the weak affinity, while structural studies demonstrate that electrostatic interactions among negatively charged amino acid residues of actin and positively charged residues of myosin are involved in the strong binding interface. We therefore conjecture that intermediate states of increasing actin–myosin engagement during the weak‐to‐strong binding transition also involve electrostatic interactions. Methods of polymer solution physics have shown that the thin actin filament can be regarded in some of its aspects as a net negatively charged polyelectrolyte. Here we employ polyelectrolyte theory to suggest how actin–myosin electrostatic interactions might be of significance in the intermediate stages of binding, ensuring an engaged power stroke of the myosin motor that transmits force to the actin filament, and preventing the motor from getting stuck in a metastable pre‐power stroke state. We provide electrostatic force estimates that are in the pN range known to operate in the cycle.     

Kinetic mechanism of myosin IXB and the contributions of two class IX-specific regions

Myosin IXb (Myo9b) was reported to be a single-headed, processive myosin. In its head domain it contains an N-terminal extension and a large loop 2 insertion that are specific for class IX myosins. We characterized the kinetic properties of purified, recombinant rat Myo9b, and we compared them with those of Myo9b mutants that had either the N-terminal extension or the loop 2 insertion deleted. Unlike other processive myosins, Myo9b exhibited a low affinity for ADP, and ADP release was not rate-limiting in the ATPase cycle. Myo9b is the first myosin for which ATP hydrolysis or an isomerization step after ATP binding is rate-limiting. Myo9b-ATP appeared to be in a conformation with a weak affinity for actin as determined by pyrene-actin fluorescence. However, in actin cosedimentation experiments, a subpopulation of Myo9b-ATP bound F-actin with a remarkably high affinity. Deletion of the N-terminal extension reduced actin affinity and increased the rate of nucleotide binding. Deletion of the loop 2 insertion reduced the actin affinity and altered the communication between actin and nucleotide-binding sites.     

Oxygen exchange between phosphate and water accompanies calcium-regulated ATPase activity of skinned fibers from rabbit skeletal muscle

The extent of oxygen exchange between phosphate and water has been measured for the calcium-regulated magnesium-dependent ATPase activity of chemically skinned fibers from rabbit skeletal muscle. The oxygen exchange was determined for isometrically held fibers by measuring with a mass spectrometer the distribution of 18O atoms in the product inorganic phosphate when ATP hydrolysis was carried out in H2(18)O. The extent of exchange was much greater in relaxed muscle (free Ca2+ less than 10(-8) M) than in calcium-activated muscle (free Ca2+ approximately equal to 3 X 10(-5) M). Activated fibers had an ATPase activity at least 30-fold greater than the relaxed fibers. These results correlate well with the extents of oxygen exchange accompanying magnesium-dependent myosin and unregulated actomyosin ATPase activities, respectively. In relaxed fibers, comparison of the amount of exchange with the ATPase activity suggests that the rate constant for the reformation of myosin-bound ATP from the myosin products complex is about 10 s-1 at 20 degrees C and pH 7.1. In each experiment the distribution of 18O in the Pi formed was incompatible with a single pathway for ATP hydrolysis. In the case of the calcium-activated fibers, the multiple pathways for ATP hydrolysis appeared to be an intrinsic property of the actomyosin ATPase in the fiber. These results indicate that in muscle fibers, as in isolated actomyosin, cleavage of protein-bound ATP is readily reversible and that association of the myosin products complex with actin promotes Pi release.