Myosin ATPase, mechanism

The simplest mechanism to explain the much faster rate of dissociation of actomyosin-S-1 by ATP than that of ATP cleavage is that actin activates the myosin ATPase by accelerating the rate at which ADP and Pj are released. That is when ATP is added to actomyosin-S-1, ATP rapidly binds and dissociates actomyosin, myosin ATPase then hydrolyzes ATP to form myosin-ADP.Pj, this state then reattaches to actin and phosphate is released much faster from actomyosin. ADP.Pj than it is from myosin.ADP.Pj, as shown in the scheme below ... 

Using two enzymes, a mammalian adenylate cyclase and myosin ATPase, as examples the application of phosphorothioate analogues to the study of the mechanism of nucleotidyl and phosphoryl transfer will be described. 

CD inhibition of actin-activated myosin ATPase at 1 1 ratio is observable only in the laboratory. Its significance as a regulatory mechanism is severely limited because the thin filaments of smooth muscle contain only 1 CD per 14 actin (Fig. 2), so that no more than 7% inhibition is possible by this mechanism. On the other hand, it has been demonstrated in numerous laboratories that CD actin ratios similar to those in vivo are potently inhibitory when tropomyosin is present. 

Marston SB, Taylor EW (1980) Comparison of the myosin and actomyosin ATPase mechanisms of the four types of vertebrate muscles. J Mol Biol 139 573600 Matsu-ura M, Ikebe M (1995) Requirement of the two -headed structure for the phosphorylation dependent regulation of smooth muscle myosin. FEBS Lett 363 246250... 

Solutions of F-actin and myosin at high ionic strength = 0.6) in vitro form a complex called actomyosin. The formation of the complex is reflected by an increase in viscosity and occurs in a deflnite molar ratio 1 molecule of myosin per 2 molecules of G-actin, the basic unit of the double-helical F-actin strand. It appears that a spike-like structure is formed, which consists of myosin molecules embedded in a backbone made of the F-actin double helix. Addition of ATP to actomyosin causes a sudden drop in viscosity due to dissociation of the complex. When this addition of ATP is followed by addition of Ca +, the myosin ATPase is activated, ATP is hydrolyzed and the actomyosin complex again restored after the ATP concentration decreases. Upon spinning of an actomyosin solution into water, flbers are obtained which, analogous to muscle flbers, contract in the presence of ATP. Glycerol extraction of muscle fibers removes all the soluble components and abolishes the semipermeability of the membrane. Such a model muscle system shows all the reactions of in vivo muscle contraction after the readdition of ATP and Ca +. This and similar model studies demonstrate that the muscle contraction mechanism is understood in principle, although some molecular details are still not clarified. 

From a practical point of view the major difference between the two approaches to enzyme kinetics, steady state and transient rate measurements, is in the concentrations of enzyme used. Steady state experiments are carried out with catalytic amounts of enzyme at concentrations negligible compared to those of the substrates or products. The rationale of transient kinetic experiments, discussed in section 5.1, will be seen to rest on the observation of complexes of enzymes with substrates and products. The importance of the direct observation and characterization of reaction intermediates for an understanding of mechanisms will be illustrated in that section. This requires enzyme concentrations sufficiently high for detection of intermediates by spectroscopic or other physical monitors. There are a number of interesting systems in vivo with enzyme and substrates at comparable concentrations and the potential kinetic consequences of such situations will be discussed in sections 5.2 and 5.3. Jencks (1989) comments in connection with a review of the transient kinetic behaviour and mechanism of one such system, the calcium pump of the sarcoplasmic reticulum, that steady state kinetics could make no contribution to an understanding of its ATPase linked reaction. The same can be said of the mechanism of myosin-ATPase, which has been elucidated in detail by transient kinetic studies (see section 5.1). 

These comments apply to all enzyme mechanisms to varying degrees and particularly also to a problem closely related to the calcium pump, that of the myosin ATPase coupled to muscle contraction. The methods used to study the latter enzyme are discussed in some detail below and are also the ones which helped to elucidate the steps involved in the coupled process of ATP hydrolysis and calcium translocation. 

Ishijima A, Kojima H, Funatsu T, Tokunaga M, Higuchi H, Tanaka H and Yanagida T 1998 Simultaneous observation of individual ATPase and mechanical events by a single myosin molcule during interaction with actin Ce//92 161-71... 

Contraction of muscle follows an increase of Ca " in the muscle cell as a result of nerve stimulation. This initiates processes which cause the proteins myosin and actin to be drawn together making the cell shorter and thicker. The return of the Ca " to its storage site, the sarcoplasmic reticulum, by an active pump mechanism allows the contracted muscle to relax (27). Calcium ion, also a factor in the release of acetylcholine on stimulation of nerve cells, influences the permeabiUty of cell membranes activates enzymes, such as adenosine triphosphatase (ATPase), Hpase, and some proteolytic enzymes and facihtates intestinal absorption of vitamin B 2 [68-19-9] (28). 

Dynein, kinesin, and myosin are motor proteins with ATPase activity that convert the chemical bond energy released by ATP hydrolysis into mechanical work. Each motor molecule reacts cyclically with a polymerized cytoskeletal filament in this chemomechanical transduction process. The motor protein first binds to the filament and then undergoes a conformational change that produces an increment of movement, known as the power stroke. The motor protein then releases its hold on the filament before reattaching at a new site to begin another cycle. Events in the mechanical cycle are believed to depend on intermediate steps in the ATPase cycle. Cytoplasmic dynein and kinesin walk (albeit in opposite... 

Even though dynein, kinesin, and myosin serve similar ATPase-dependent chemomechanical functions and have structural similarities, they do not appear to be related to each other in molecular terms. Their similarity lies in the overall shape of the molecule, which is composed of a pair of globular heads that bind microtubules and a fan-shaped tail piece (not present in myosin) that is suspected to carry the attachment site for membranous vesicles and other cytoplasmic components transported by MT. The cytoplasmic and axonemal dyneins are similar in structure (Hirokawa et al., 1989 Holzbaur and Vallee, 1994). Current studies on mutant phenotypes are likely to lead to a better understanding of the cellular roles of molecular motor proteins and their mechanisms of action (Endow and Titus, 1992). 

The myosins are a superfamily of proteins that have the ability to convert energy released by ATP is hydrolysis into mechanical work. There are many forms of myosin, all of which have ATPase activity and an actin-binding site that is located... 

In summary, therefore, solution and fiber biochemistry have provided some idea about how ATP is used by actomyosin to generate force. Currently, it seems most likely that phosphate release, and also an isomerization between two AM.ADP.Pj states, are closely linked to force generation in muscle. ATP binds rapidly to actomyosin (A.M.) and is subsequently rapidly hydrolyzed by myosin/actomyosin. There is also a rapid equilibrium between M. ADP.Pj and A.M.ADP.Pj (this can also be seen in fibers from mechanical measurements at low ionic strength). The rate limiting step in the ATPase cycle is therefore likely to be release of Pj from A.M.ADP.Pj, in fibers as well as in solution, and this supports the idea that phosphate release is associated with force generation in muscle. 

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