Loop 1 of transducer region in mammalian class I myosin, Myo1b, modulates actin affinity, ATPase activity, and nucleotide access

Loop 1, a flexible surface loop in the myosin motor domain, comprises in part the transducer region that lies near the nucleotide-binding site and is proposed from structural studies to be responsible for the kinetic tuning of product release following ATP hydrolysis (1). Biochemical studies have shown that loop 1 affects the affinity of actin-myosin-II for ADP, motility and the V(max) of the actin-activated Mg2+-ATPase activity, possibly through P(i) release (2-8). To test the influence of loop 1 on the mammalian class I myosin, Myo1b, chimeric molecules in which (i) loop 1 of a truncated form of Myo1b, Myo1b1IQ, was replaced with either loop 1 from other myosins; (ii) loop 1 was replaced with glycine; or (iii) some amino acids in the loop were substituted with alanine and were expressed in baculovirus, and their interactions with actin and nucleotide were evaluated. The steady-state actin-activated ATPase activity; rate of ATP-induced dissociation of actin from Myo1b1IQ; rate of ADP release from actin-Myo1b1IQ; and the affinity of actin for Myo1b1IQ and Myo1b1IQ.ADP differed in the chimeras versus wild type, indicating that loop 1 has a much wider range of effects on the coupling between actin and nucleotide binding events than previously thought. In particular, the biphasic ATP-induced dissociation of actin from actin-Myo1b1IQ was significantly altered in the chimeras. This provided evidence that loop 1 contributes to the accessibility of the nucleotide pocket and is involved in the integration of information from the actin-, nucleotide-, gamma-P(i)-, and calmodulin-binding sites and predicts that loop 1 modulates the load dependence of the motor.     

Mechanism of regulation of Acanthamoeba myosin-IC by heavy-chain phosphorylation

The ATPase activity of myosin-Is from lower eukaryotes is activated by phosphorylation by the p21-activated kinase family at the TEDS site on an actin-binding surface-loop. This actin-binding loop is the site of a cardiac myosin-II mutation responsible for some forms of familial hypertrophic cardiomyopathy. To determine the mechanism of myosin-I regulation by heavy-chain phosphorylation (HCP) and to better understand the importance of this loop in the function of all myosin isoforms, we performed a kinetic investigation of the regulatory mechanism of the Acanthamoeba myosin-IC motor domain (MIC(IQ)). Phosphorylated and dephosphorylated MIC(IQ) show actin-activated ATPase activity; however, HCP increases the ATPase activity >20-fold. HCP does not greatly affect the rate of phosphate release from MIC in the absence of actin, as determined by single turnover experiments. Additionally, HCP does not significantly affect the affinity of myosin for actin in the absence or presence of ATP, the rate of ATP-induced dissociation of actoMIC(IQ), the affinity of ADP, or the rate of ADP release. Sequential-mix single-turnover experiments show that HCP regulates the rate of phosphate release from actin-bound MIC(IQ). We propose that the TEDS-containing actin-binding loop plays a direct role in regulating phosphate release and the force-generating (A-to-R) transition of myosin-IC.     

Disturbed communication between actin- and nucleotide-binding sites in a myosin II with truncated 50/20-kDa junction.

The kinetic and functional consequences of deleting nine residues from an actin-binding surface loop (loop 2) were examined to investigate the role of this region in myosin function. The nucleotide binding properties of myosin were not altered by the deletion. However, the deletion affected actin binding and the communication between the actin- and nucleotide-binding sites. The affinity of M765NL for actin (644 nM) was approximately 100-fold lower than that of wild-type construct M765 (5.8 nM). Despite this reduction in affinity, actin binding weakened the affinity of ADP for the motor to a similar extent for both mutant and wild-type constructs. The addition of 0.5 microM actin decreased ADP affinity from 0.6 to 34 microM for M765NL and from 1.6 to 39 microM for M765. In contrast, communication between the actin- and nucleotide-binding sites appears disturbed in regard to phosphate release: thus, basal ATPase activity for M765NL (0.19 s-1) was 3-fold larger than for M765 (0.06 s-1), and the stimulation of ATPase activity by actin was 5-fold lower for M765NL. These results indicate different paths of communication between the actin- and nucleotide-binding sites, in regard to ADP and Pi release, and they confirm that loop 2 is involved in high affinity actin binding.     

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.     

Magnesium regulates ADP dissociation from myosin V

Processivity in myosin V is mediated through the mechanical strain that results when both heads bind strongly to an actin filament, and this strain regulates the timing of ADP release. However, what is not known is which steps that lead to ADP release are affected by this mechanical strain. Answering this question will require determining which of the several potential pathways myosin V takes in the process of ADP release and how actin influences the kinetics of these pathways. We have addressed this issue by examining how magnesium regulates the kinetics of ADP release from myosin V and actomyosin V. Our data support a model in which actin accelerates the release of ADP from myosin V by reducing the magnesium affinity of a myosin V-MgADP intermediate. This is likely a consequence of the structural changes that actin induces in myosin to release phosphate. This effect on magnesium affinity provides a plausible explanation for how mechanical strain can alter this actin-induced acceleration. For actomyosin V, magnesium release follows phosphate release and precedes ADP release. Increasing magnesium concentration to within the physiological range would thus slow both the ATPase activity and the velocity of movement of this motor.     

Kinetic characterization of a monomeric unconventional myosin V construct.

An expressed, monomeric murine myosin V construct composed of the motor domain and two calmodulin-binding IQ motifs (MD(2IQ)) was used to assess the regulatory and kinetic properties of this unconventional myosin. In EGTA, the actin-activated ATPase activity of MD(2IQ) was 7.4 +/- 1.6 s(-1) with a K(app) of approximately 1 microM (37 degrees C), and the velocity of actin movement was approximately 0.3 micrometer/s (30 degrees C). Calcium inhibited both of these activities, but the addition of calmodulin restored the values to approximately 70% of control, indicating that calmodulin dissociation caused inhibition. In contrast to myosin II, MD(2IQ) is highly associated with actin at physiological ionic strength in the presence of ATP, but the motor is in a weakly bound conformation based on the pyrene-actin signal. The rate of dissociation of acto-MD(2IQ) by ATP is fast (>850 s(-1)), and ATP hydrolysis occurs at approximately 200 s(-1). The affinity of acto-MD(2IQ) for ADP is somewhat higher than that of smooth S1, and ADP dissociates more slowly. Actin does not cause a large increase in the rate of ADP release, nor does the presence of ADP appreciably alter the affinity of MD(2IQ) for actin. These kinetic data suggest that monomeric myosin V is not processive.     

The sequence of the myosin 50-20K loop affects Myosin's affinity for actin throughout the actin-myosin ATPase cycle and its maximum ATPase activity

We are interested in the role that solvent-exposed, proteolytically sensitive surface loops play in myosin function. The 25-50K loop, or loop 1, is near the ATP binding site, while the 50-20K loop (loop 2) is in the actin binding site. Through chimeric studies, we have found that loop 1 affects ADP release [Murphy, C. T., and Spudich, J. A. (1998) Biochemistry 37, 6738-44], while loop 2 affects the actin-activated ATPase activity [Uyeda, T. Q.-P., et al. (1994) Nature 368, 567-9]. In the study described here, we have found that the kcat of the actin-activated ATPase activity is changed by the loop 2 substitutions in a manner that reflects the relative actin-activated ATPase activities of the donor myosins. Additionally, changes in loop 2 affect the affinity of myosin for actin both in the presence and in the absence of nucleotides. Pre-steady-state studies together with the ATPase and affinity data suggest that while loop 2 does not affect interactions between myosin and nucleotide, it plays a role in determining the affinity of myosin for actin in various nucleotide states and in the rate-limiting transition allowing phosphate release.     

The kinetic mechanism of Myo1e (human myosin-IC)

Myo1e is the widely expressed subclass-1 member of the myosin-I family. We performed a kinetic analysis of a truncated myo1e that consists of the motor and the single IQ motif with a bound calmodulin. We determined the rates and equilibrium constants for the key steps in the ATPase cycle. The maximum actin activated ATPase rate (V(max)) and the actin concentration at half-maximum of V(max) (K(ATPase)) of myo1e are similar to those of the native protein. The K(ATPase) is low (approximately 1 microm), however the affinity of myo1e for actin in the presence of ATP is very weak. A weak actin affinity and a rapid rate of phosphate release result in a pathway under in vitro assay conditions in which phosphate is released while myo1e is dissociated from actin. Actin activation of the ATPase activity and the low K(ATPase) are the result of actin activation of ADP release. We propose that myo1e is tuned to function in regions of high concentrations of cross-linked actin filaments. Additionally, we found that ADP release from actomyo1e is > 10-fold faster than other vertebrate myosin-I isoforms. We propose that subclass-1 myosin-Is are tuned for rapid sliding, whereas subclass-2 isoforms are tuned for tension maintenance or stress sensing.     

Characterization of the motor activity of mammalian myosin VIIA

Myosin VIIA was cloned from rat kidney, and the construct (M7IQ5) containing the motor domain, IQ domain, and the coiled-coil domain as well as the full-length myosin VIIA (M7full) was expressed. The M7IQ5 contained five calmodulins. Based upon native gel electrophoresis and gel filtration, it was found that M7IQ5 was single-headed, whereas M7full was two-headed, suggesting that the tail domain contributes to form the two-headed structure. M7IQ5 had Mg(2+)-ATPase activity that was markedly activated by actin with K(actin) of 33 microm and V(max) of 0.53 s(-1) head(-1). Myosin VIIA required an extremely high ATP concentration for ATPase activity, ATP-induced dissociation from actin, and in vitro actin-translocating activity. ADP markedly inhibited the actin-activated ATPase activity. ADP also significantly inhibited the ATP-induced dissociation of myosin VIIA from actin. Consistently, ADP decreased K(actin) of the actin-activated ATPase. ADP decreased the actin gliding velocity, although ADP did not stop the actin gliding even at high concentration. These results suggest that myosin VIIA has slow ATP binding or low affinity for ATP and relatively high affinity for ADP. The directionality of myosin VIIA was determined by using the polarity-marked dual fluorescence-labeled actin filaments. It was found that myosin VIIA is a plus-directed motor.     

Addition of lysines to the 50/20 kDa junction of myosin strengthens weak binding to actin without affecting the maximum ATPase activity

Much interest has centered on two surface loops in the motor domain to explain the differences in enzymatic and mechanical properties of myosin isoforms. We showed that two invariant lysines at the C-terminal end of loop 2, which is part of the actin-binding interface, are required to obtain actin activation [Joel et al. (2001) J. Biol. Chem. 276, 2998-3003]. Here we investigate the effects of increasing positive charge in the variable portion of loop 2 of smooth muscle heavy meromyosin (smHMM). Increasing the net positive charge by +4 increased the affinity for actin in the presence and absence of ATP. The K(m) for actin-activated ATPase activity decreased 15-fold, but V(max) was unchanged, showing that "weak binding" of myosin for actin can be significantly strengthened without increasing the rate-limiting step for V(max). The mutant HMM had slower rates of in vitro motility and ADP release compared to WT HMM. ADP release and motility, which were both salt-dependent, correlated linearly with each other. Loop 2 thus plays a major role in setting the affinity for actin but also affects ADP release and motility. Because the actin- and nucleotide-binding regions communicate, mutations to one region can impact multiple facets of myosin's mechanical and enzymatic properties.     

Human myosin Vc is a low duty ratio, nonprocessive molecular motor

Myosin Vc is the product of one of the three genes of the class V myosin found in vertebrates. It is widely found in secretory and glandular tissues, with a possible involvement in transferrin trafficking. Transient and steady-state kinetic studies of human myosin Vc were performed using a truncated, single-headed construct. Steady-state actin-activated ATPase measurements revealed a V(max) of 1.8 +/- 0.3 s(-1) and a K(ATPase) of 43 +/- 11 microm. Unlike previously studied vertebrate myosin Vs, the rate-limiting step in the actomyosin Vc ATPase pathway is the release of inorganic phosphate (~1.5 s(-1)), rather than the ADP release step (~12.0-16.0 s(-1)). Nevertheless, the ADP affinity of actomyosin Vc (K(d) = 0.25 +/- 0.02 microm) reflects a higher ADP affinity than seen in other myosin V isoforms. Using the measured kinetic rates, the calculated duty ratio of myosin Vc was approximately 10%, indicating that myosin Vc spends the majority of the actomyosin ATPase cycle in weak actin-binding states, unlike the other vertebrate myosin V isoforms. Consistent with this, a fluorescently labeled double-headed heavy meromyosin form showed no processive movements along actin filaments in a single molecule assay, but it did move actin filaments at a velocity of approximately 24 nm/s in ensemble assays. Kinetic simulations reveal that the high ADP affinity of actomyosin Vc may lead to elevations of the duty ratio of myosin Vc to as high as 64% under possible physiological ADP concentrations. This, in turn, may possibly imply a regulatory mechanism that may be sensitive to moderate changes in ADP concentration.     

Kinetic characterization of the function of myosin loop 4 in the actin-myosin interaction

Myosin interacts with actin during its enzymatic cycle, and actin stimulates myosin's ATPase activity. There are extensive interaction surfaces on both actin and myosin. Several surface loops of myosin play different roles in actomyosin interaction. However, the functional role of loop 4 in actin binding is still ambiguous. We explored the role of loop 4 by either mutating its conserved acidic group, Glu-365, to Gln (E365Q), or by replacing the entire loop with three glycines (DeltaAL) in a Dictyostelium discoideum myosin II motor domain (MD) containing a single tryptophan residue. This native tryptophan (Trp-501) is located in the relay loop and is sensitive to nucleotide binding and lever-arm movement. Fluorescence and fast kinetic measurements showed that the mutations in loop 4 do not alter the enzymatic steps of the ATPase cycle in the absence of actin. By contrast, actin binding was significantly weakened in the absence and presence of ADP and ATP in both mutants. Because the strength of actin-myosin interaction increases in the order of rigor, ADP, and ATP complex, we conclude that loop 4 is a functional actin-binding region that stabilizes actomyosin complex, particularly in weak actin-binding states.     

Human myosin III is a motor having an extremely high affinity for actin

Myosin IIIA is expressed in photoreceptor cells and thought to play a critical role in phototransduction processes, yet its function on a molecular basis is largely unknown. Here we clarified the kinetic mechanism of the ATPase cycle of human myosin IIIA. The steady-state ATPase activity was markedly activated approximately 10-fold with very low actin concentration. The rate of ADP off from actomyosin IIIA was 10 times greater than the overall cycling rate, thus not a rate-determining step. The rate constant of the ATP hydrolysis step of the actin-dissociated form was very slow, but the rate was markedly accelerated by actin binding. The dissociation constant of the ATP-bound form of myosin IIIA from actin is submicromolar, which agrees well with the low K(actin). These results indicate that ATP hydrolysis predominantly takes place in the actin-bound form for actomyosin IIIA ATPase reaction. The obtained K(actin) was much lower than the previously reported one, and we found that the autophosphorylation of myosin IIIA dramatically increased the K(actin), whereas the V(max) was unchanged. Our kinetic model indicates that both the actin-attached hydrolysis and the P(i) release steps determine the overall cycle rate of the dephosphorylated form. Although the stable steady-state intermediates of actomyosin IIIA ATPase reaction are not typical strong actin-binding intermediates, the affinity of the stable intermediates for actin is much higher than conventional weak actin binding forms. The present results suggest that myosin IIIA can spend a majority of its ATP hydrolysis cycling time on actin.     

Kinetic mechanism of human myosin IIIA

Myosin IIIA is specifically expressed in photoreceptors and cochlea and is important for the phototransduction and hearing processes. In addition, myosin IIIA contains a unique N-terminal kinase domain and C-terminal tail actin-binding motif. We examined the kinetic properties of baculovirus expressed human myosin IIIA containing the kinase, motor, and two IQ domains. The maximum actin-activated ATPase rate is relatively slow (k(cat) = 0.77 +/- 0.08 s(-1)), and high actin concentrations are required to fully activate the ATPase rate (K(ATPase) = 34 +/- 11 microm). However, actin co-sedimentation assays suggest that myosin III has a relatively high steady-state affinity for actin in the presence of ATP (K(actin) approximately 7 microm). The rate of ATP binding to the motor domain is quite slow both in the presence and absence of actin (K(1)k(+2) = 0.020 and 0.001 microm(-1).s(-1), respectively). The rate of actin-activated phosphate release is more than 100-fold faster (85 s(-1)) than the k(cat), whereas ADP release in the presence of actin follows a two-step mechanism (7.0 and 0.6 s(-1)). Thus, our data suggest a transition between two actomyosin-ADP states is the rate-limiting step in the actomyosin III ATPase cycle. Our data also suggest the myosin III motor spends a large fraction of its cycle in an actomyosin ADP state that has an intermediate affinity for actin (K(d) approximately 5 microm). The long lived actomyosin-ADP state may be important for the ability of myosin III to function as a cellular transporter and actin cross-linker in the actin bundles of sensory cells.     

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.     

Mechanism of inhibition of skeletal muscle actomyosin by N-benzyl-p-toluenesulfonamide

N-Benzyl-p-toluenesulfonamide (BTS) is a small organic molecule that specifically inhibits the contraction of fast skeletal muscle fibers. To determine the mechanism of inhibition by BTS, we performed a kinetic analysis of its effects on the elementary steps of the actomyosin subfragment-1 ATPase cycle. BTS decreases the steady-state acto-S1 ATPase rate approximately 10-fold and increases the actin concentration for half-maximal activation. BTS primarily affects three of the elementary steps of the reaction pathway. It decreases the rate of P(i) release >20-fold in the absence of actin and >100-fold in the presence of actin. It decreases the rate of S1.ADP dissociation from 3.9 to 0.8 s(-)(1) while decreasing the S1.ADP dissociation constant from 2.3 to 0.8 microM. BTS weakens the apparent affinity of S1.ADP for actin, increasing the K(d) from 7.0 to 29.5 microM. ATP binding to S1, hydrolysis, and the affinity of nucleotide-free S1 for actin are unaffected by BTS. Kinetic modeling indicates that the binding of BTS to myosin depends on actin association/dissociation and on nucleotide state. Our results suggest that the reduction of the acto-S1 ATPase rate is due to the inhibition of P(i) release, and the suppression of tension is due to inhibition of P(i) release in conjunction with the decreased apparent affinity of S1.ADP.P(i) and S1.ADP for actin.     

Dynamics of the upper 50-kDa domain of myosin V examined with fluorescence resonance energy transfer

The upper 50-kDa region of myosin may be critical for coupling between the nucleotide- and actin-binding regions. We introduced a tetracysteine motif in the upper 50-kDa domain (residues 292-297) of myosin V containing a single IQ domain (MV 1IQ), allowing us to label this site with the fluorescein biarscenical hairpin-binding dye (FlAsH) (MV 1IQ FlAsH). The enzymatic properties of MV 1IQ FlAsH were similar to those of unlabeled MV 1IQ except for a 3-fold reduced ADP-release rate. MV 1IQ FlAsH was also capable of moving actin filaments in the in vitro motility assay. To examine rotation of the upper 50-kDa region, we determined the difference in the degree of energy transfer from N-methylanthraniloyl (mant)-labeled nucleotides to FlAsH in both steady-state and transient kinetic experiments. The energy transfer efficiency was higher with mant-ATP (0.65 +/- 0.02) compared with mant-ADP (0.55 +/- 0.02) in the absence of actin. Stopped-flow measurements suggested that the energy transfer efficiency decreased with phosphate release (0.04 s(-1)) in the absence of actin. In contrast, upon mixing MV 1IQ FlAsH in the ADP.P(i) state with actin, a decrease in the energy transfer signal was observed at a rate of 13 s(-1), similar to the ADP release rate. Our results demonstrate there was no change in the energy transfer signal upon actin-activated phosphate release and suggest that actin binding alters the dynamics of the upper 50-kDa region, which may be critical for the ability of myosin to bind tightly to both ADP and actin.     

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.     

Magnesium, ADP, and actin binding linkage of myosin V: evidence for multiple myosin V-ADP and actomyosin V-ADP states

The [Mg(2+)] dependence of ADP binding to myosin V and actomyosin V was measured from the fluorescence of mantADP. Time courses of MgmantADP dissociation from myosin V and actomyosin V are biphasic with fast observed rate constants that depend on the [Mg(2+)] and slow observed rate constants that are [Mg(2+)]-independent. Two myosin V-MgADP states that are in reversible equilibrium, one that exchanges nucleotide and cation slowly (strong binding) and one that exchanges nucleotide and cation rapidly (weak binding), account for the data. The two myosin V-MgADP states are of comparable energies, as indicated by the relatively equimolar partitioning at saturating magnesium. Actin binding lowers the affinity for bound Mg(2+) 2-fold but shifts the isomerization equilibrium approximately 6-fold to the weak ADP binding state, lowering the affinity and accelerating the overall rate of MgADP release. Actin does not weaken the affinity or accelerate the release of cation-free ADP, indicating that actin and ADP binding linkage is magnesium-dependent. Myosin V and myosin V-ADP binding to actin was assayed from the quenching of pyrene actin fluorescence. Time courses of myosin V-ADP binding and release are biphasic, consistent with the existence of two (weak and strong) quenched pyrene actomyosin V-ADP conformations. We favor a sequential mechanism for actomyosin V dissociation with a transition from strong to weak actin-binding conformations preceding dissociation. The data provide evidence for multiple myosin-ADP and actomyosin-ADP states and establish a kinetic and thermodynamic framework for defining the magnesium-dependent coupling between the actin and nucleotide binding sites of myosin.     

Two Drosophila myosin transducer mutants with distinct cardiomyopathies have divergent ADP and actin affinities

Two Drosophila myosin II point mutations (D45 and Mhc(5)) generate Drosophila cardiac phenotypes that are similar to dilated or restrictive human cardiomyopathies. Our homology models suggest that the mutations (A261T in D45, G200D in Mhc(5)) could stabilize (D45) or destabilize (Mhc(5)) loop 1 of myosin, a region known to influence ADP release. To gain insight into the molecular mechanism that causes the cardiomyopathic phenotypes to develop, we determined whether the kinetic properties of the mutant molecules have been altered. We used myosin subfragment 1 (S1) carrying either of the two mutations (S1(A261T) and S1(G200D)) from the indirect flight muscles of Drosophila. The kinetic data show that the two point mutations have an opposite effect on the enzymatic activity of S1. S1(A261T) is less active (reduced ATPase, higher ADP affinity for S1 and actomyosin subfragment 1 (actin · S1), and reduced ATP-induced dissociation of actin · S1), whereas S1(G200D) shows increased enzymatic activity (enhanced ATPase, reduced ADP affinity for both S1 and actin · S1). The opposite changes in the myosin properties are consistent with the induced cardiac phenotypes for S1(A261T) (dilated) and S1(G200D) (restrictive). Our results provide novel insights into the molecular mechanisms that cause different cardiomyopathy phenotypes for these mutants. In addition, we report that S1(A261T) weakens the affinity of S1 · ADP for actin, whereas S1(G200D) increases it. This may account for the suppression (A261T) or enhancement (G200D) of the skeletal muscle hypercontraction phenotype induced by the troponin I held-up(2) mutation in Drosophila.