The reversal of the myosin and actomyosin ATPase reactions and the free energy of ATP binding to myosin

Evidence is presented that both myosin and actomyosin in presence of Mg²⁺ and KCl catalyze an incorporation of ³²P_i into ATP. The rate with actomyosin is about $\text{1}\text{500}$ the rate of ATP hydrolysis; the rate with myosin is less than $\text{1}\text{100}$ of that with actomyosin. With myosin, but not with actomyosin, an apparent initial “burst” of ³²P_i incorporation into ATP is observed. Actin binding thus promotes ATP dissociation. The data with myosin allow estimation of both the amount of enzyme-bound [³²P]-ATP present and the rate constant, k_?1, for dissociation of the myosin· ATP. From these results and other data a ?ΔG^o for ATP binding to myosin of 12–13 kcal/mole may be estimated, with a much lower ?ΔG^o for hydrolysis of enzyme-bound ATP. Protein conformational change accompanying ATP binding appears to be the principal means of capture of energy from the overall reaction of ATP cleavage.     

Enzymatic studies on the skeletal myosin A and actomyosin of aging rats.

Myosin A and actomyosin were isolated from the skeletal muscle of old and young rats. The velocity of the Ca2+ activated myosin A ATPase was increased in the case of the older animals. On the other hand the velocity of the Mg-2plus activated actomyosin ATPase was decreased in the skeletal muscle of the aging rats. At 5 X 10-5M EGTA concentration the inhibition of the Mg-2plus activated myosin B ATPase of the 1-month-old rats was two- to threefold smaller than that of the older animals. It was shown that the myosin A component of the actomyosin was responsible for the decreased troponin inhibition in the case of the 1-month-old rats. Between the ages of 1 month and 29 months the number of free myosin A SH groups decreases by 50%. The lipid peroxidation in the muscle of the 1-month-old animals.     

A classical and ab initio study of the interaction of the myosin triphosphate binding domain with ATP.

We used classical molecular mechanics (MM) simulations and quantum mechanical (QM) structural relaxations to examine the active site of myosin when bound to ATP. Two conformations of myosin have been determined by x-ray crystallography. In one, there is no direct interaction between switch 2 and the nucleotide (open state). In the other (closed state), the universally conserved switch 2 glycine forms a hydrogen bond with a gamma-phosphate oxygen. MM simulations indicate that the two states are thermodynamically stable and allow us to investigate the extent to which the P-loop, switch 1, and switch 2 are involved in hydrolysis. We find that the open structure has a higher affinity for ATP than the closed structure, and that ATP is distorted toward a transition state by interactions with the protein. We also examine how the structure of the binding site changes with either MgATP or CaATP as the nucleotide in myosin in the open conformer. Our analyses suggest that higher CaATPase rates occur because the leaving phosphate (P(i)) group is more weakly bound and dissociation occurs faster. Finally, we validate the use of a particular formulation of a QM methodology (Car-Parrinello) to further refine the structures of the active site.     

The reaction between actomyosin and adenosine triphosphate

1. A study is made of the effect of adenosine triphosphate (ATP) upon the viscosity of solutions of actomyosin in 0.5 M KCl. 2. The observed effects are discussed in terms of an initial drop of the viscosity (viscosity response) and its subsequent slow reversal (recovery effect). The latter is ascribed to a decrease in the ATP concentration through enzymatic hydrolysis. 3. The recovery effect is inhibited by Mg, activated by Ca, in accordance with the effect of these ions on the activity of myosin-ATPase. 4. The viscosity response is not inhibited, probably promoted by Mg. It is not promoted, probably inhibited by Ca. 5. The viscosity response is induced not only by ATP, but to a certain extent also by inosinetriphosphate, inorganic triphosphate, and inorganic pyrophosphate, not by adenosine diphosphate or monophosphate. 6. The viscosity response could be obtained with enzymatically inactive myosin. 7. It is concluded that the effect of ATP upon myosin does not depend on its enzymatic hydrolysis.     

Structure and function of the two heads of the myosin molecule. IV. Physiological functions of various reaction intermediates in myosin adenosinetriphosphatase, studied by the interaction between actomyosin and 8-bromoadenosine triphosphate.

The kinetic properties of the hydrolyses of 8-Br ATP and 8-SCH3 ATP by myosin [EC 3.6.1.3] and actomyosin were compared with those of ATP, and the following results were obtained. The Ca-NTPase activities of myosin using these two ATP analogs as substrates were smaller than that of ATPase, and the NTPase activities toward these analogs were strongly suppressed by EDTA. The Mg-NTPase activities toward these analogs were higher in a medium of high ionic strength than in a medium of low ionic strength, in contrast to the activity of Mg-ATPase. These analogs did not produce any initial burst of Pi liberation, activation of myosin NTPase by F-actin, or superprecipitation of actomyosin. The interactions between 8-Br ATP and HMM, acto-HMM, actomyosin, and myofibrils were studied in detail in the presence of Mg2+ in medium of low ionic strength. The Michaelis constant, Km, and the maximum rate, Vm, of 8-Br ATPase of HMM were 27 muM and 21 min-1, respectively. The fluorescence change of HMM induced by 8-Br ATP also followed the Michaelis-Menten equation, and the Michaelis constant, Kf1, was as low as 4 muM. Acto-HMM and acto-S-1 were fully dissociated by the addition of 8-Br ATP. The relation between the extent of dissociation of acto-HMM and the concentration of 8-Br ATP followed the Michaelis-Menten equation, and the apparent dissociation constant, Kd, was 22 muM. This Kd value is almost equal to the Km value of 8-Br ATPase of HMM described above. Myofibrillar contraction was not supported by 8-Br ATP. It was concluded that in the myosin NTPase reaction with 8-Br ATP as a substrate, M2NTP but not MNDPP is formed in route (1), while MNTP is formed in route (2). It was also concluded that the key intermediate for the actomyosin NTPase reaction is MNDPP, and that dissociation of acto-HMM is induced by the formation of M2NTP and MNTP in routes (1) and (2), respectively.