Rate-limiting step in the actomyosin adenosinetriphosphatase cycle: studies with myosin subfragment 1 cross-linked to actin

Although there is agreement that actomyosin can hydrolyze ATP without dissociation of the actin from myosin, there is still controversy about the nature of the rate-limiting step in the ATPase cycle. Two models, which differ in their rate-limiting step, can account for the kinetic data. In the four-state model, which has four states containing bound ATP or ADP . Pi, the rate-limiting step is ATP hydrolysis (A . M . ATP in equilibrium A . M . ADP . Pi). In the six-state model, which we previously proposed, the rate-limiting step is a conformational change which occurs before Pi release but after ATP hydrolysis. A difference between these models is that only the four-state model predicts that almost no acto-subfragment 1 (S-1) . ADP . Pi complex will be formed when ATP is mixed with acto . S-1. In the present study, we determined the amount of acto . S-1 . ADP . Pi formed when ATP is mixed with S-1 cross-linked to actin [Mornet, D., Bertrand, R., Pantel, P., Audemard, E., & Kassab, R. (1981) Nature (London) 292, 301-306]. The amount of acto . S-1 . ADP . Pi was determined both from intrinsic fluorescence enhancement and from direct measurement of Pi. We found that at mu = 0.013 M, the fluorescence magnitude in the presence of ATP of the cross-linked actin . S-1 preparation was about 50% of the value obtained with S-1, while at mu = 0.053 M the fluorescence magnitude was about 70% of that obtained with S-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

ATPase and shortening rates in frog fast skeletal myofibrils by time-resolved measurements of protein-bound and free Pi.

Shortening and ATPase rates were measured in Ca2+-activated myofibrils from frog fast muscles in unloaded conditions at 4 degrees C. ATPase rates were determined using the phosphate-binding protein method (free phosphate) and quench flow (total phosphate). Shortening rates at near zero load (V0) were estimated by quenching reaction mixtures 50 ms to 10 s old at pH 3.5 and measuring sarcomere lengths under the optical microscope. As with the rabbit psoas myofibrils (C. Lionne, F. Travers, and T. Barman, 1996, Biophys. J. 70:887-895), the ATPase progress curves had three phases: a transient Pi burst, a fast linear phase (kF), and a deceleration to a slow phase (kS). Evidence is given that kF is the ATPase rate of shortening myofibrils. V0 is in good agreement with mechanical measurements in myofibrils and fibers. Under the same conditions and at saturation in ATP, V0 and kF are 2.4 microm half-sarcomere(-1) s(-1) and 4.6 s(-1), and their Km values are 33 and 200 microM, respectively. These parameters are higher than found with rabbit psoas myofibrils. The myofibrillar kF is higher than the fiber ATPase rates obtained previously in frog fast muscles but considerably lower than obtained in skinned fibers by the phosphate-binding protein method (Z. H. He, R. K. Chillingworth, M. Brune, J. E. T. Corrie, D. R. Trentham, M. R. Webb, and M. R. Ferenczi, 1997, J. Physiol. 50:125-148). We show that, with frog as with rabbit myofibrillar ATPase, phosphate release is the rate-limiting step.     

Mechanochemical coupling in muscle: attempts to measure simultaneously shortening and ATPase rates in myofibrils.

We studied the ATPase of shortening myofibrils at 4 degrees C by the rapid flow quench method. The progress curve has three phases: a P(i) burst, a fast linear phase kF of duration tB, and a deceleration to a slow kS. We propose that kF is the ATPase of myofibrils shortening under zero external load; at tB shortening and ATPase rates are reduced by passive resistance. The total ATP consumed during the rapid shortening is ATPc. Our purpose was to obtain information on the myofibrillar shortening velocity from their ATPase progress curves. We tested tB as an indicator of shortening velocity by determining the effects of different probes upon it and the other ATPase parameters. The dependence of tB upon the initial sarcomere length was linear, giving a shortening velocity close to that of muscle fibres (Vo). The Km of ATP was larger for tB than for kF, as found with fibers for Vo and their ATPase. ADP and 2,3-butanedione monoxime, but not P(i), inhibited tB to the same extent as Vo. The delta H for tB and Vo were similar. ATPc was independent of the sarcomere length, implying that the more the myofibrils shorten, the less ATP expended per myosin head per micron shortened. We propose that tB can be used as an indicator for myofibrillar shortening velocities.     

Myosin Va becomes a low duty ratio motor in the inhibited form

Vertebrate myosin Va is a typical processive motor with high duty ratio. Recent studies have revealed that the actin-activated ATPase activity of the full-length myosin Va (M5aFull) is inhibited at a low [Ca(2+)], which is due to the formation of a folded conformation of M5aFull. To clarify the underlying inhibitory mechanism, we analyzed the actin-activated ATP hydrolysis mechanism of the M5aFull at the inhibited and the activated states, respectively. Marked differences were found in the hydrolysis, P(i) release, and ADP release steps between the activated and the inhibited states. The kinetic constants of these steps of the activated state were similar to those of the unregulated S1 construct, in which the rate-limiting step was the ADP release step. On the other hand, the P(i) release rate from acto-M5aFull was decreased in EGTA by >1,000-fold, which makes this step the rate-limiting step for the actin-activated ATP hydrolysis cycle of M5aFull. The ADP off rate from acto-M5aFull was decreased by approximately 10-fold, and the equilibrium between the prehydrolysis state and the post hydrolysis state was shifted toward the former state in the inhibited state of M5aFull. Because of these changes, M5aFull spends a majority of the ATP hydrolysis cycling time in the weak actin binding state. The present results indicate that M5aFull molecules at a low [Ca(2+)] is inhibited as a cargo transporter not only due to the decrease in the cross-bridge cycling rate but also due to the decrease in the duty ratio thus being dissociated from actin.     

Strain-dependent cross-bridge cycle for muscle. II. Steady-state behavior.

Quantitative predictions of steady-state muscle properties from the strain-dependent cross-bridge for muscle are presented. With a stiffness of 5.4 x 10(-4) N/m per head, a throw distance of 11 nm, and three allowed actin sites/head, isometric properties and their dependence on phosphate and nucleotide levels are well described if the tension-generating step occurs before phosphate release. At very low ATP levels, rigorlike states with negative strain are predicted. The rate-limiting step for cycling and ATP consumption is strain-blocked ADP release for isometric and slowly shortening muscle. Under rapid shortening, ATP hydrolysis on detached heads is the rate-limiting step, and the ratio of bound ATP to bound ADP.Pi increases by a factor of 7. At large positive strains, bound heads must be forcibly detached from actin to account for tension in rapid extension, but forced detachment in shortening has no effect without destroying isometric attached states. Strain-blocked phosphate release as proposed produces modest inhibition of the ATPase rate under rapid shortening, sufficient to give a maximum for one actin site per helix turn. Alternative cross-bridge models are discussed in the light of these predictions.     

The effect of polyethylene glycol on the mechanics and ATPase activity of active muscle fibers

We have used polyethylene glycol (PEG) to perturb the actomyosin interaction in active skinned muscle fibers. PEG is known to potentiate protein-protein interactions, including the binding of myosin to actin. The addition of 5% w/v PEG (MW 300 or 4000) to active fibers increased fiber tension and decreased shortening velocity and ATPase activity, all by 25-40%. Variation in [ADP] or [ATP] showed that the addition of PEG had little effect on the dissociation of the cross-bridge at the end of the power stroke. Myosin complexed with ADP and the phosphate analog V(i) or AlF(4) binds weakly to actin and is an analog of a pre-power-stroke state. PEG substantially enhances binding of these states both in active fibers and in solution. Titration of force with increasing [P(i)] showed that PEG increased the free energy available to drive the power stroke by about the same amount as it increased the free energy available from the formation of the actomyosin bond. Thus PEG potentiates the binding of myosin to actin in active fibers, and it provides a method for enhancing populations of some states for structural or mechanical studies, particularly those of the normally weakly bound transient states that precede the power stroke.     

Cryoenzymic studies on an organized system: myofibrillar ATPases and shortening.

We have exploited cryoenzymology, first, to probe the product release steps of myofibrillar ATPase under relaxing conditions and, second, to define the conditions for studying the contractile process in slow motion. Cryoenzymology implies perturbation by temperature and by the antifreeze added to allow for work at subzero temperatures. Here, we studied myofibrillar shortening and ATPases by the rapid quench flow method over a wide temperature range (-15 to 30 degrees C) in two antifreezes, 40% ethylene glycol and 20% methanol. The choice of solvent and temperature was dictated by the purpose of the experiment. Ethylene glycol (40%) is suitable for investigating the kinetics of the products release steps which is difficult in water. In this cryosolvent, the myofibrillar ATPase is not activated by Ca2+ nor is there shortening, except under special conditions, i.e., Ca2+ plus strong rigor bridges [Stehle, R., Lionne, C., Travers, F., and Barman, T. (1998) J. Muscl. Res. Cell Motil. 19, 381-392]. By the use of the glycol, we show that at low Ca2+ the kinetics of the ADP release are much faster with myofibrils than with S1. On the other hand, the kinetics of the Pi release were very similar for the two materials. Therefore, we suggest that, upon Ca2+ activation, only the Pi release kinetics are accelerated. In 20% methanol, in the presence of Ca2+, myofibrils shortened at temperatures above -2 degrees C but not below. At a given temperature above -2 degrees C, both the shortening and ATPase rates were reduced by the methanol. The temperature dependences of the myofibrillar ATPases (+/-Ca2+) converged with a decrease in temperature: at 20 degrees C, Ca2+ activated 30-fold, but at -15 degrees C, only about 5-fold. We suggest that studies in methanol may open the way for an investigation of muscle contraction in slow motion and, further, to obtain thermodynamic information on the internal forces involved in the shortening process.     

Evidence for increased low force cross-bridge population in shortening skinned skeletal muscle fibers: implications for actomyosin kinetics.

The dynamic characteristics of the low force myosin cross-bridges were determined in fully calcium-activated skinned rabbit psoas muscle fibers shortening under constant loads (0.04-0.7 x full isometric tension Po). The shortening was interrupted at various times by a ramp stretch (duration, 10 ms; amplitude, up to 1.8% fiber length) and the resulting tension response was recorded. Except for the earlier period of velocity transients, the tension response showed nonlinear dependence on stretch amplitude; i.e., the magnitude of the tension response started to rise disproportionately as the stretch exceeded a critical amplitude, as in the presence of inorganic phosphate (Pi). This result, as well as the result of stiffness measurement, suggests that the low force cross-bridges similar to those observed in the presence of Pi (presumably A.M.ADP.Pi) are significantly populated during shortening. The critical amplitude of the shortening fibers was greater than that of isometrically contracting fibers, suggesting that the low force cross-bridges are more negatively strained during shortening. As the load was reduced from 0.3 to 0.04 P0, the shortening velocity increased more than twofold, but the amount of the negative strain stayed remarkably constant (approximately 3 nm). This This insensitiveness of the negative strain to velocity is best explained if the dissociation of the low force cross-bridges is accelerated approximately in proportion to velocity. Along with previous reports, the results suggest that the actomyosin ATPase cycle in muscle fibers has at least two key reaction steps in which rate constants are sensitively regulated by shortening velocity and that one of them is the dissociation of the low force A.M.ADP.Pi cross-bridges. This step may virtually limit the rate of actomyosin ATPase turnover and help increase efficiency in fibers shortening at high velocities.     

Butanedione monoxime suppresses contraction and ATPase activity of rabbit skeletal muscle

The effects of 2,3-butanedione 2-monoxime (BDM) on mechanical responses of glycerinated fibers and the ATPase activity of heavy meromyosin (HMM) and myofibrils have been studied using rabbit skeletal muscle. The mechanical responses and the ATPase activity were measured in similar conditions (ionic strength 0.06-0.2 M, 0.4-4 mM MgATP, 0-20 mM BDM, 2-20 degrees C and pH 7.0). BDM reversibly reduced the isometric tension, shortening speed, and instantaneous stiffness of the fibers. BDM also inhibited myofibrillar and HMM ATPase activities. The inhibitory effect on the relative ATPase activity of HMM was not influenced by the addition of actin or troponin-tropomyosin-actin. High temperature and low ionic strength weakened BDM's suppression of contraction of the fibers and the ATPase activity of contracting myofibrils, but not of the HMM, acto-HMM and relaxed myofibrillar ATPase activity. The size of the initial phosphate burst at 20 degrees C was independent of the concentration of BDM. These results suggest that the suppression of contraction of muscle fibers is due mainly to direct action of BDM on the myosin molecules.     

Effect of 2,3-butanedione monoxime on myosin and myofibrillar ATPases. An example of an uncompetitive inhibitor.

2,3-Butanedione monoxime (BDM) reversibly inhibits force production in muscle. At least part of its action appears to be directly on the contractile apparatus. To understand better its mechanism of action, we studied the effect of BDM on the steps of myosin subfragment 1 Mg(2+)-ATPase in 0.1 M potassium acetate, pH 7.4. Because of the rapidity of certain processes, we experimented at 4 degrees C and our main technique was the rapid flow quench method. By varying the experimental conditions (relative concentrations of reagents, time scale, quenching agent), it was possible to study selectively the different steps of the S1 Mg(2+)-ATPase: [formula: see text] At saturation (20 mM), BDM had two major effects on the ATPase. First, it increased the equilibrium constant of the cleavage step (K3) from 2 to > 10. Second, it slowed the kinetics of the release of Pi by an order of magnitude (k4; from 0.054 to 0.004 s-1). By contrast, the kinetics of the binding of ATP (k) and the release of ADP (k6) were little affected by BDM. Thus, the oxime appears to interact specifically with M**.ADP.Pi, and it is a rare example of an uncompetitive inhibitor. Its effect is to reduce the steady-state concentration of the "strong" actin binding state M*.ADP and to increase that of the "weak" binding state, M**.ADP.Pi. The effect of BDM on the initial ATPase of Ca2+ activated myofibrils was very similar to that on S1 ATPase. Thus, with myofibrils too BDM seems to exert its main effect subsequent to the initial binding and cleavage steps.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of myofibrillar and actomyosin subfragment 1 adenosinetriphosphatase by adenosine 5'-diphosphate, pyrophosphate, and adenyl-5'-yl imidodiphosphate

Adenosine 5'-diphosphate (ADP), inorganic pyrophosphate (PPi), and adenyl-5'-yl imidodiphosphate (AMPPNP) act as competitive inhibitors of the ATPase of myofibrils and actomyosin subfragment 1 (acto-S1). At I = 0.2 M, pH 7, and 15 degrees C, the inhibition constants for rabbit myofibrils are 0.17, 3, and 5 mM, respectively; the values for frog myofibrils at 0 degrees C are very similar, being 0.22, 1.5, and 2.5 mM. The inhibition constant of AMPPNP is about 2 orders of magnitude larger than the reported dissociation constant for fibers [Marston, S. B., Rodger, C. D., & Tregear, R. T. (1976) J. Mol. Biol. 104, 263-276]. A possible reason for this difference is that AMPPNP binding results in the dissociation of one head of each myosin molecule. The inhibition constants for rabbit acto-S1 cross-linked with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide measured under the same conditions were 0.12, 2.6, and 3.5 mM for ADP, PPi, and AMPPNP, respectively. The inhibition of cross-linked and native acto-S1 was compared at low ionic strength and was found to be similar. The value for ADP is very similar to reported values of the dissociation constant whereas the inhibition constants for AMPPNP and PPi are an order of magnitude weaker [Greene, L. E., & Eisenberg, E. (1980) J. Biol. Chem. 255, 543-548].     

The mechanism of smooth muscle caldesmon-tropomyosin inhibition of the elementary steps of the actomyosin ATPase

Caldesmon is a component of smooth muscle thin filaments that inhibits the actomyosin ATPase via its interaction with actin-tropomyosin. We have performed a comprehensive transient kinetic characterization of the actomyosin ATPase in the presence of smooth muscle caldesmon and tropomyosin. At physiological ratios of caldesmon to actin (1 caldesmon/7 actin monomers) actomyosin ATPase is inhibited by about 75%. Inhibitory caldesmon concentrations had little effect upon the rate of S1 binding to actin, actin-S1 dissociation by ATP, and dissociation of ADP from actin-S1 x ADP; however the rate of phosphate release from the actin-S1 x ADP x P(i) complex was decreased by more than 80%. In addition the transient of phosphate release displayed a lag of up to 200 ms. The presence of a lag phase indicates that a step on the pathway prior to phosphate release has become rate-limiting. Premixing the actin-tropomyosin filaments with myosin heads resulted in the disappearance of the lag phase. We conclude that caldesmon inhibition of the rate of phosphate release is caused by the thin filament being switched by caldesmon to an inactive state. The active and inactive states correspond to the open and closed states observed in skeletal muscle thin filaments with no evidence for the existence of a third, blocked state. Taken together these data suggest that at physiological concentrations, caldesmon controls the isomerization of the weak binding complex to the strong binding complex, and this causes the inhibition of the rate of phosphate release. This inhibition is sufficient to account for the inhibition of the steady state actomyosin ATPase by caldesmon and tropomyosin.     

ADP inhibition of myosin V ATPase activity

The kinetic mechanism of myosin V is of great interest because recent evidence indicates that the two-headed myosin V molecule functions as a processive motor, i.e., myosin V is capable of moving along an actin filament for many catalytic cycles of the motor without dissociating. Three recent publications assessing the kinetics of single-headed myosin V provide different conclusions regarding the mechanism, particularly the rate-limiting step of the cycle. One study (, Proc. Natl. Acad. Sci. USA. 96:13726-13731) identifies ADP release as the rate-limiting step and provides a kinetic explanation for myosin V processivity. The others (, J. Biol. Chem. 274:27448-27456;, J. Biol. Chem. 275:4329-4335) do not identify the rate-limiting step but conclude that it is not ADP release. We show experimental and simulated data demonstrating that the inconsistencies in the reports may be due to difficulties in the measurement of the steady-state ATPase rate. Under standard assay conditions, ADP competes with ATP, resulting in product inhibition of the ATPase rate. This presents technical problems in analyzing and interpreting the kinetics of myosin V and likely of other members of the myosin family with high ADP affinities.     

Structure and function of the two heads of the myosin molecule. I. Binding of adenosine diphosphate to myofibrils during the adenosinetriphosphatase reaction.

1. The myosin content of myofibrils was found to be 51% by SDS-gel electrophoresis. 2. The initial burst of Pi liberation of the ATPase [EC 3.6.1.3] of a solution of myofibrils in 1 M KCl was measured in 0.5 M KCl, and found to be 0.93 mole/mole of myosin. 3. The amount of ADP bound to myofibrils during the ATPase reaction and the ATPase activity were measured by coupling the myofibrillar ATPase reaction with sufficient amounts of pyruvate kinase [EC 2.7.1.40] and PEP to regenerate ATP. The maximum amount of ADP bound to myofibrils in 0.05M KCl and in the relaxed state was about 1.5 mole/mole of myosin. On the other hand, the ATPase activity exhibited substrate inhibition, and the amount of ATP required for a constant level of ATPase activity was smaller than that required for the maximum binding of ADP to myofibrils. 4. The maximum amount of ADP bound to myofibrils in 0.5 M KCl was about 1.9 mole/mole of myosin. When about one mole of ADP was found to 1 mole of myosin in myofibrils, the myofibrillar ATPase activity reached the saturated level, and with further increase in the concentration of ATP one more mole of ADP was found per mole of myosin.     

Functional role of loop 2 in myosin V

Myosin V is molecular motor that is capable of moving processively along actin filaments. The kinetics of monomeric myosin V containing a single IQ domain (MV 1IQ) differ from nonprocessive myosin II in that actin affinity is higher, phosphate release is extremely rapid, and ADP release is rate-limiting. We generated two mutants of myosin V by altering loop 2, a surface loop in the actin-binding region thought to alter actin affinity and phosphate release in myosin II, to determine the role that this loop plays in the kinetic tuning of myosin V. The loop 2 mutants altered the apparent affinity for actin (K(ATPase)) without altering the maximum ATPase rate (V(MAX)). Transient kinetic analysis determined that the rate of binding to actin, as well as the affinity for actin, was dependent on the net positive charge of loop 2, while other steps in the ATPase cycle were unchanged. The maximum rate of phosphate release was unchanged, but the affinity for actin in the M.ADP.Pi-state was dramatically altered by the mutations in loop 2. Thus, loop 2 is important for allowing myosin V to bind to actin with a relatively high affinity in the weak binding states but does not play a direct role in the product release steps. The ability to maintain a high affinity for actin in the weak binding states may prevent diffusion away from the actin filament and increase the degree of processive motion of myosin V.     

Actin and light chain isoform dependence of myosin V kinetics

Recent studies on myosin V report a number of kinetic differences that may be attributed to the different heavy chain (chicken vs mouse) and light chain (essential light chains vs calmodulin) isoforms used. Understanding the extent to which individual light chain isoforms contribute to the kinetic behavior of myosin V is of critical importance, since it is unclear which light chains are bound to myosin V in cells. In addition, all studies to date have used alpha-skeletal muscle actin, whereas myosin V is in nonmuscle cells expressing beta- and gamma-actin. Therefore, we characterized the actin and light chain dependence of single-headed myosin V kinetics. The maximum actin-activated steady-state ATPase rate (V(max)) of a myosin V construct consisting of the motor domain and first light chain binding domain is the same when either of two essential light chain isoforms or calmodulin is bound. However, with bound calmodulin, the K(ATPase) is significantly higher and there is a reduction in the rate and equilibrium constants for ATP hydrolysis, indicating that the essential light chain favors formation of the M. ADP.P(i) state. No kinetic parameters of myosin V are strongly influenced by the actin isoform. ADP release from the actin-myosin complex is the rate-limiting step in the ATPase cycle with all actin and light chain isoforms. We postulate that although there are significant light-chain-dependent alterations in the kinetics that could affect myosin V processivity in in vitro assays, these differences likely are minimized under physiological conditions.     

Kinetic mechanism and regulation of myosin VI.

Myosin VI is the only pointed end-directed myosin identified and is likely regulated by heavy chain phosphorylation (HCP) at the actin-binding site in vivo. We undertook a detailed kinetic analysis of the actomyosin VI ATPase cycle to determine whether there are unique adaptations to support reverse directionality and to determine the molecular basis of regulation by HCP. ADP release is the rate-limiting step in the cycle. ATP binds slowly and with low affinity. At physiological nucleotide concentrations, myosin VI is strongly bound to actin and populates the nucleotide-free (rigor) and ADP-bound states. Therefore, myosin VI is a high duty ratio motor adapted for maintaining tension and has potential to be processive. A mutant mimicking HCP increases the rate of P(i) release, which lowers the K(ATPase) but does not affect ADP release. These measurements are the first to directly measure the steps regulated by HCP for any myosin. Measurements with double-headed myosin VI demonstrate that the heads are not independent, and the native dimer hydrolyzes multiple ATPs per diffusional encounter with an actin filament. We propose an alternating site model for the stepping and processivity of two-headed high duty ratio myosins.     

Structural characterization of the binding of Myosin*ADP*Pi to actin in permeabilized rabbit psoas muscle

When myosin is attached to actin in a muscle cell, various structures in the filaments are formed. The two strongly bound states (A*M*ADP and A*M) and the weakly bound A*M*ATP states are reasonably well understood. The orientation of the strongly bound myosin heads is uniform ("stereospecific" attachment), and the attached heads exhibit little spatial fluctuation. In the prehydrolysis weakly bound A*M*ATP state, the orientations of the attached myosin heads assume a wide range of azimuthal and axial angles, indicating considerable flexibility in the myosin head. The structure of the other weakly bound state, A*M*ADP*P(i), however, is poorly understood. This state is thought to be the critical pre-power-stroke state, poised to make the transition to the strongly binding, force-generating states, and hence it is of particular interest for understanding the mechanism of contraction. However, because of the low affinity between myosin and actin in the A*M*ADP*P(i) state, the structure of this state has eluded determination both in isolated form and in muscle cells. With the knowledge recently gained in the structures of the weakly binding M*ATP, M*ADP*P(i) states and the weakly attached A*M*ATP state in muscle fibers, it is now feasible to delineate the in vivo structure of the attached state of A*M*ADP*P(i). The series of experiments presented in this article were carried out under relaxing conditions at 25 degrees C, where approximately 95% of the myosin heads in the skinned rabbit psoas muscle contain the hydrolysis products. The affinity for actin is enhanced by adding polyethylene glycol (PEG) or by lowering the ionic strength in the bathing solution. Solution kinetics and binding constants were determined in the presence and in the absence of PEG. When the binding between actin and myosin was increased, both the myosin layer lines and the actin layer lines increased in intensity, but the intensity profiles did not change. The configuration (mode) of attachment in the A*M*ADP*P(i) state is thus unique among the intermediate attached states of the cross-bridge ATP hydrolysis cycle. One of the simplest explanations is that both myosin filaments and actin filaments are stabilized (e.g., undergo reduced spatial fluctuations) by the attachment. The alignment of the myosin heads in the thick filaments and the alignment of the actin monomers in the thin filaments are improved as a result. The compact atomic structure of M*ADP*P(i) with strongly coupled domains may contribute to the unique attachment configuration: the "primed" myosin heads may function as "transient struts" when attached to the thin filaments.     

Correlation between steady-state ATP hydrolysis and vanadate-induced ADP trapping in Human P-glycoprotein. Evidence for ADP release as the rate-limiting step in the catalytic cycle and its modulation by substrates

P-glycoprotein (Pgp) is a transmembrane protein conferring multidrug resistance to cells by extruding a variety of amphipathic cytotoxic agents using energy from ATP hydrolysis. The objective of this study was to understand how substrates affect the catalytic cycle of ATP hydrolysis by Pgp. The ATPase activity of purified and reconstituted recombinant human Pgp was measured using a continuous cycling assay. Pgp hydrolyzes ATP in the absence of drug at a basal rate of 0.5 micromol x min x mg(-1) with a K(m) for ATP of 0.33 mm. This basal rate can be either increased or decreased depending on the Pgp substrate used, without an effect on the K(m) for ATP or 8-azidoATP and K(i) for ADP, suggesting that substrates do not affect nucleotide binding to Pgp. Although inhibitors of Pgp activity, cyclosporin A, its analog PSC833, and rapamycin decrease the rate of ATP hydrolysis with respect to the basal rate, they do not completely inhibit the activity. Therefore, these drugs can be classified as substrates. Vanadate (Vi)-induced trapping of [alpha-(32)P]8-azidoADP was used to probe the effect of substrates on the transition state of the ATP hydrolysis reaction. The K(m) for [alpha-(32)P]8-azidoATP (20 microm) is decreased in the presence of Vi; however, it is not changed by drugs such as verapamil or cyclosporin A. Strikingly, the extent of Vi-induced [alpha-(32)P]8-azidoADP trapping correlates directly with the fold stimulation of ATPase activity at steady state. Furthermore, P(i) exhibits very low affinity for Pgp (K(i) approximately 30 mm for Vi-induced 8-azidoADP trapping). In aggregate, these data demonstrate that the release of Vi trapped [alpha-(32)P]8-azidoADP from Pgp is the rate-limiting step in the steady-state reaction. We suggest that substrates modulate the rate of ATPase activity of Pgp by controlling the rate of dissociation of ADP following ATP hydrolysis and that ADP release is the rate-limiting step in the normal catalytic cycle of Pgp.     

Functional divergence of human cytoplasmic myosin II: kinetic characterization of the non-muscle IIA isoform

Cytoplasmic (or non-muscle) myosin II isoforms are widely expressed molecular motors playing essential cellular roles in cytokinesis and cortical tension maintenance. Two of the three human non-muscle myosin II isoforms (IIA and IIB) have been investigated at the protein level. Transient kinetics of non-muscle myosin IIB showed that this motor has a very high actomyosin ADP affinity and slow ADP release. Here we report the kinetic characterization of the non-muscle myosin IIA isoform. Similar to non-muscle myosin IIB, non-muscle myosin IIA shows high ADP affinity and little enhancement of the ADP release rate by actin. The ADP release rate constant, however, is more than an order of magnitude higher than the steady-state ATPase rate. This implies that non-muscle myosin IIA spends only a small fraction of its ATPase cycle time in strongly actin-bound states, which is in contrast to non-muscle myosin IIB. Non-muscle myosin II isoforms thus appear to have distinct enzymatic properties that may be of importance in carrying out their cellular functions.