The mechanism of skeletal muscle myosin ATPase. The mechanism of ADP interaction

The mechanism of interaction between ADP and the myosin active center has been studied using a transient kinetic technique. The results show that the interaction of ADP with the myosin active center is a homogeneous process independent of the association state of the active centers; namely, whether ADP interacts with the monomeric myosin subfragment-1, or with the dimeric forms heavy meromyosin and myosin. The kinetics of the interaction conforms to a simple two-step reaction mechanism for ADP binding. The kinetic and thermodynamic constants for this mechanism have been determined. In addition, analysis of the binding isotherm indicates that the two active sites in heavy meromyosin and myosin function as identical and independent sites.     

The mechanism of skeletal muscle myosin ATPase. Interaction of myosin active center with ATP and with ADP

The kinetics of the fluorescence enhancement and the transient release of H+ caused by the binding of ADP to the active center of myosin has been compared to that caused by myosin-ATP interaction. The results show that both the time courses of the fluorescence enhancement and the transient H+ release caused by ADP binding, like that caused by ATP hydrolysis in the initial burst, are monophasic exponential processes. The fact that the rates of these two processes are also equal suggests that they both reflect the same mechanistic event in the mechanism of ADP binding. The kinetics of ADP binding as measured by the fluorescence enhancement and the H+ release is different from that of ATP. This is in agreement with our previous finding that the enhancement of fluorescence and the transient release of H+, in the case of ATP, reflect the initial burst of ATP hydrolysis, whereas in the case of ADP, they represent a conformational change in the myosin-ADP complex. The magnitude of the H+ transient caused by the initial burst is approximately equal to that caused by ADP binding. The amplitude of the fluorescence enhancement caused by ADP binding is equal to one-third of that caused by the initial burst.     

A model for the active site of skeletal muscle myosin.

A model for a main element of the active site of skeletal muscle myosin is presented that relates directly to the 92 amino acid fragment (p₁₀) of myosin recently described by Elzinga &; Collins (1977). In this model, the substrate, an eight-membered cyclic complex of MgATP, fits tightly into a 16 amino acid segment of p₁₀ and interacts with seven of its amino acids. A main feature of the model is the important role played by the one molecule of N^τ-methylhistidine² that is present in each myosin heavy chain. At the site, it is postulated that this rare amino acid functions as a donor ligand to Mg²⁺. Once N^τ-methylhistidine is put in place next to the metal, the other amino acids that appear to form a pocket come easily into position around the MgATP. These amino acids with their postulated functions are: tyrosine 72, which through a Mg-bound water, or perhaps directly, is attached to the Mg; histidine 76, which donates a proton to the P_γ of ATP; lysine 78, which binds electrostatically to P_β of ATP; phenylalanines 80 and 81, which flank the purine ring of ATP; and aspartate 66, which forms a hydrogen bond to the 6-amino group of adenine. The Mg-coordination role ascribed to N^τ-methylhistidine 69 in skeletal muscle myosin could be taken by histidine 69 in cardiac myosin and in other muscle myosins that do not contain the methylated amino acid.The choice of p₁₀ to contain a main element of the active site is based on: (a) the presence in p₁₀ of the essential sulfhydryl groups, SH1 and SH2, whose modification affects the ATPase activity of myosin; (b) the presence in ρ₁₀ of N^τ-methylhistidine, an unusual amino acid whose methylation in skeletal muscle we take as an indicator for a special function at the active site; (c) the position of p₁₀ in the primary structure near the junction between subfragment 1 and subfragment 2 (the hinge region) where, we postulate, enzymatic events at the active site are coupled to movements of the hinge that occur during contraction; (d) indications that the DTNB light chain, probably involved in regulation, is also near the hinge; (e) the effects of MgATP at the active site on the chemical reactivity of three SH groups (SH1, SH2 and SH3) located near the hinge; and (f) the effect of hinge cleavage on the oxygen exchange reaction catalyzed at the active site. The correlation of all these observations forms the basis for our placement of part of the active site on p₁₀ near the subfragment 1-subfragment 2 hinge.     

The role of the myosin ATPase activity in adaptive thermogenesis by skeletal muscle

Resting skeletal muscle is a major contributor to adaptive thermogenesis, i.e., the thermogenesis that changes in response to exposure to cold or to overfeeding. The identification of the "furnace" that is responsible for increased heat generation in resting muscle has been the subject of a number of investigations. A new state of myosin, the super relaxed state (SRX), with a very slow ATP turnover rate has recently been observed in skeletal muscle (Stewart et al. in Proc Natl Acad Sci USA 107:430-435, 2010). Inhibition of the myosin ATPase activity in the SRX was suggested to be caused by binding of the myosin head to the core of the thick filament in a structural motif identified earlier by electron microscopy. To be compatible with the basal metabolic rate observed in vivo for resting muscle, most myosin heads would have to be in the SRX. Modulation of the population of this state, relative to the normal relaxed state, was proposed to be a major contributor to adaptive thermogenesis in resting muscle. Transfer of only 20% of myosin heads from the SRX into the normal relaxed state would cause muscle thermogenesis to double. Phosphorylation of the myosin regulatory light chain was shown to transfer myosin heads from the SRX into the relaxed state, which would increase thermogenesis. In particular, thermogenesis by myosin has been proposed to play a role in the dissipation of calories during overfeeding. Up-regulation of muscle thermogenesis by pharmaceuticals that target the SRX would provide new approaches to the treatment of obesity or high blood sugar levels.     

Comparison of kinetic properties of the ATPase reaction of arterial smooth muscle myosin with skeletal muscle myosin

Myosin was prepared from arterial smooth muscle, and a hybrid actomyosin was formed from arterial myosin and rabbit skeletal muscle F-actin. We performed kinetics on the ATPase reaction [EC 3.6.1.3] of arterial myosin and the hybrid actomyosin at high ionic strength, and compared the kinetic properties of arterial myosin ATPase with those of skeletal muscle myosin ATPase. No significant difference was found between these two myosins in the size of the initial Pi burst, the amount of bound nucleotides, and the rates of various elementary steps in the ATPase reaction. On the other hand, two important differences were observed between the hybrid actomyosin and skeletal muscle actomyosin: (i) The amounts of ATP necessary for complete dissociation of the hybrid and skeletal muscle actomyosins were 2 and 1 mol/mol of myosin, respectively. (ii) The rate of dissociation of the hybrid actomyosin induced by ATP was much lower than that of skeletal muscle actomyosin and also was lower than that of fluorescence enhancement.     

Modification of cardiac and smooth muscle myosins with 2,4,6-trinitrobenzenesulfonate. Evidence for differences in structure around the active sites of cardiac, smooth, and skeletal muscle myosin ATPase.

Myosins purified from cardiac (porcine heart) and smooth (chicken gizzard) muscles were modified with 2,4,6-trinitrobenzenesulfonate (TNBS) and the effects on the kinetic properties of myosin ATPase [EC 3.6.1.3] were studied. The following results were obtained. 1. About 0.5 mol of TNBS per mol of myosin head was incorporated rapidly, irrespective of the presence of PP1 (2mM), into both types of myosin studied. 2. The size of the initial burst of P1 liberation for both myosins was found to be 0.5--0.6 mol/mol head. 3. The rapid incorporation of TNBS into cardiac muscle myosin was accompanied by a rapid decrease in the size of the initial P1 burst, and it was completely lost after modification for 20 min. However, smooth muscle myosin retained its P1 burst. 4. The EDTA (K+)-ATPase activity of both myosins modified in the presence or absence of PP1 decreased sharply with incorporation of TNBS. 5. Superprecipitation and ATPase activity of reconstituted actomyosin from cardiac myosin and skeletal F-actin decreased only after 10 min of modification with TNBS in the absence of PP1. 6. The spectra of TNP bound to myosins from cardiac and smooth muscles were unchanged by the addition of PP1. The above findings are compared with those previously obtained for skeletal muscle myosin [Miyanishi, T., Inoue, A., & Tonomura, Y. (1979) J. Biochem. 85, 747--753], and the structural and functional differences among the myosins derived from skeletal, cardiac, and smooth muscles are discussed.     

The mechanism of the skeletal muscle myosin ATPase. II. Relationship between the fluorescence enhancement induced by ATP and the initial Pi burst.

A major question about the mechanism of the myosin ATPase is how much of the fluorescence change which accompanies the binding of ATP to myosin is due to the conformational change induced by ATP and how much is due to the subsequent hydrolysis of ATP in the initial Pi burst. Several laboratories have suggested that the maximal rate of the fluorescence change represents the rate of the irreversible conformational change induced by ATP. In the present study, the rate of irreversible ATP binding, the rate of the initial Pi burst, and the rate of the fluorescence enhancement were compared under varied conditions. The results show that: 1) the fluorescence enhancement is mainly due to the hydrolysis of ATP in the initial Pi burst rather than to the conformational change induced by the binding of ATP; 2) the rate of the initial Pi burst is considerably slower than the rate of irreversible ATP binding at high ATP concentration; 3) the rate of the initial Pi burst is almost the same as the rate of the fluorescence enhancement. Therefore, the maximum rate of the fluorescence enhancement represents the rate of the initial Pi burst rather than the rate of the conformational change induced by ATP binding.     

Characterization of the active site of platelet myosin in comparison to smooth and skeletal muscle myosin.

Heavy meromyosin subfragment-1 from human platelets and chicken gizzard exhibited an identical chromatographic pattern on agarose-ATP columns both in the absence and in the presence of Ca²⁺ and Mg²⁺. In the presence of Ca²⁺, the behavior differed from that of rabbit white skeletal muscle subfragment-1. The reaction of lysyl residues of platelet myosin with 2,4,6-trinitrobenzene sulfonate did not affect the K⁺- or Mg²⁺-stimulated ATPase activity. A similar behavior was exhibited by chicken gizzard myosin whereas trinitrophenylation of the more active lysyl residues in skeletal muscle myosin caused a marked increase in Mg²⁺-stimulated and a decrease in K⁺-stimulated ATPase activity. These features may point to a similar location of the essential lysyl residue in platelet and smooth muscle myosin, which is different from that of skeletal muscle. Alkylation of thiol groups by N-ethyl maleimide in the absence of added nucleotides resulted in a loss of K⁺-ATPase and in an increase in the Ca²⁺-ATPase in all three myosins, the increase for the skeletal myosin being much greater than for the platelet and chicken gizzard preparations. Alkylation of myosin in the presence of MgADP led to a decrease in K⁺-ATPase of all preparations whereas the Ca²⁺-ATPase as a function of time exhibited a maximum for the platelet and skeletal muscle proteins. These features may point to a certain similarity with respect to the active site of platelet and smooth muscle myosins and a difference between these and skeletal muscle myosin.     

Cholinergic sites in skeletal muscle. I. Denervation effects.

Cholinergic interactions in systems derived from rat skeletal mixed muscle are detailed. The isotherms of the binding of [125I]diiodo-alpha-bungarotoxin over the range of 10(-10)-10(-5) M toxin have been separated into a "nonspecific" component exclusive to the toxin and a "specific" component that binds both the toxin and d-tubocurarine. The "specific" component appears to reflect two independent sets of binding sites. One of the sets has an affinity constant on the order of 10(9) M-1. Following denervation, the number of sites in this high-affinity set begins to increase after 3 days, reaches a peak (28-fold higher than normal) on the 8th day, and begins to decline. Similar results are obtained when sensitivity of this set to an antibody derived from patients with myasthenia gravis is examined. This sensitivity is reflected by the inhibition of the alpha-bungarotoxin binding by the myasthenic IgG fraction. Following denervation, sensitivity first appears on day 3 progresses coincidentally with the increase in new sites in the set. The charcteristics of this set suggest that it represents the acetylcholine receptor and that the new sites appearing during the course of denervation are extrajunctional receptor sites. The interaction with the myasthenic IgG indicates an antigenic difference between junctional and extrajunctional receptors. The second set of specific binding sites has an affinity constant on the order of 10(5) M-1. The number of sites in this set increases only fivefold as a result of denervation. The increase also begins between days 2 and 3. The definition of this low affinity set of sites is not presently clear.     

The influence of ethylenediaminetetraacetate on white skeletal muscle myosin.

Myosin from rabbit white skeletal muscle was treated with 10 mM EDTA in 150 mM phosphate buffer. After precipitation of myosin by dialysis against a 14-fold volume of water, EDTA-treated myosin, myosin before treatment and the supernatant from the treatment of myosin with EDTA were examined on sodium dodecyl sulphate-polyacrylamide gels by electrophoresis. It has been found that the quantity of LC2 light chains diminished after treatment with EDTA, and the supernatant contained the LC2 light chains. Treatment of myosin with EDTA in the presence of Mg2+ does not change the stoichiometry of the LC2 light chain and the supernatant is free from LC2 light chains. The treatment of myosin with p-chloromercuri-benzoate leads to dissociation of the same amount of LC2 light chains. It is suggested that divalent cations and thiol groups are engaged in the attachment of LC2 light chain to the myosin molecule.     

The mechanism of the skeletal muscle myosin ATPase. III. Relationship of the H+ release and the protein absorbance change induced by ATP to the initial Pi burst.

Several phenomena are associated with the binding of ATP to myosin: 1) a fluorescence enhancement, 2) a release of H+, and 3) a protein absorbance change. In the accompanying paper (Chock, S. P., Chock, P. B., and Eisenberg, E. (1979) J. Biol. Chem. 254, 3236-3243), it was demonstrated that the fluorescence enhancement is mainly caused by the hydrolysis of ATP in the initial Pi burst rather than by the conformational change induced by the irreversible binding of ATP. In the present study, the cause of the H+ release and the protein absorbance change were investigated. The results show that like the rate of the fluorescence enhancement the rates of the H+ release and the protein absorbance change level off at high ATP concentration at a much lower rate than the rate of irreversible ATP binding. Furthermore, under all conditions tested, the rates of the H+ release and the protein absorbance change are equal to the rate of the initial Pi burst. Therefore, like the fluorescence enhancement, most of the H+ release and the protein absorbance change are associated with the initial Pi burst rather than the binding of ATP.     

Nucleotide regulatory sites on skeletal myosin

Myosin Ca2+-ATPase activity decreased in the presence of ADP. Free ATP acted either as an activator or as an inhibitor depending on its concentration. The inhibition caused by ADP or ATP followed a competitive pattern with respect to the substrate. ATP, at activating concentrations, competed with dinitrophenol and with the anions SCN-, CN- and HCO3- for the same binding sites of myosin, whereas ADP did not compete with them. These results suggest that the nucleotide regulatory site or sites, different from the hydrolytic sites, seem to coincide with the anion binding sites.     

Effect of staphylococcus active substances on ATPase activity of smooth muscle actomyosin and myosin

The effect of staphylococcus active substances--protein A (PA) and peptidoglican (PG) at concentrations 10(-6)-10(-2) mg/ml on the ATPase activity of pig stomach natural actomyosin and myosin was studied. It was shown that PA and PG at direct contact with smooth muscle contractile proteins caused the activation and inhibition of ATPase activity, respectively. On the basis of this investigation it was assumed that staphylococcal active substances were able to modify of the ATPase activity smooth muscle contractile proteins perhaps via direct action on the myosin molecule, which could be accompanied by conformational changes of the active center of myosin ATPase.