Release Of Hydrolysis Products Research Articles

Force and number of myosin motors during muscle shortening and the coupling with the release of the ATP hydrolysis products.

Muscle contraction is due to cyclical ATP-driven working strokes in the myosin motors while attached to the actin filament. Each working stroke is accompanied by the release of the hydrolysis products, orthophosphate and ADP. The rate of myosin-actin interactions increases with the increase in shortening velocity. We used fast half-sarcomere mechanics on skinned muscle fibres to determine the relation between shortening velocity and the number and strain of myosin motors and the effect of orthophosphate concentration. A model simulation of the myosin-actin reaction explains the results assuming that orthophosphate and then ADP are released with rates that increase as the motor progresses through the working stroke. The ADP release rate further increases by one order of magnitude with the rise of negative strain in the final motor conformation. These results provide the molecular explanation of the relation between the rate of energy liberation and shortening velocity during muscle contraction. The chemo-mechanical cycle of the myosin II--actin reaction in situ has been investigated in Ca(2+)-activated skinned fibres from rabbit psoas, by determining the number and strain (s) of myosin motors interacting during steady shortening at different velocities (V) and the effect of raising inorganic phosphate (Pi) concentration. It was found that in control conditions (no added Pi ), shortening at V ≤ 350 nm s(-1) per half-sarcomere, corresponding to force (T) greater than half the isometric force (T0 ), decreases the number of myosin motors in proportion to the reduction of T, so that s remains practically constant and similar to the T0 value independent of V. At higher V the number of motors decreases less than in proportion to T, so that s progressively decreases. Raising Pi concentration by 10 mM, which reduces T0 and the number of motors by 40-50%, does not influence the dependence on V of number and strain. A model simulation of the myosin-actin reaction in which the structural transitions responsible for the myosin working stroke and the release of the hydrolysis products are orthogonal explains the results assuming that Pi and then ADP are released with rates that increase as the motor progresses through the working stroke. The rate of ADP release from the conformation at the end of the working stroke is also strain-sensitive, further increasing by one order of magnitude within a few nanometres of negative strain. These results provide the molecular explanation of the relation between the rate of energy liberation and the load during muscle contraction.

Mechanism of substrate translocation by a ring-shaped ATPase motor at millisecond resolution.

Ring-shaped, hexameric ATPase motors fulfill key functions in cellular processes, such as genome replication, transcription, or protein degradation, by translocating a long substrate through their central pore powered by ATP hydrolysis. Despite intense research efforts, the atomic-level mechanism transmitting chemical energy from hydrolysis into mechanical force that translocates the substrate is still unclear. Here we employ all-atom molecular dynamics simulations combined with advanced path sampling techniques and milestoning analysis to characterize how mRNA substrate is translocated by an exemplary homohexameric motor, the transcription termination factor Rho. We find that the release of hydrolysis product (ADP + Pi) triggers the force-generating process of Rho through a 0.1 millisecond-long conformational transition, the time scale seen also in experiment. The calculated free energy profiles and kinetics show that Rho unidirectionally translocates the single-stranded RNA substrate via a population shift of the conformational states of Rho; upon hydrolysis product release, the most favorable conformation shifts from the pretranslocation state to the post-translocation state. Via two previously unidentified intermediate states, the RNA chain is seen to be pulled by six K326 side chains, whose motions are induced by highly coordinated relative translation and rotation of Rho's six subunits. The present study not only reveals in new detail the mechanism employed by ring-shaped ATPase motors, for example the use of loosely bound and tightly bound hydrolysis reactant and product states to coordinate motor action, but also provides an effective approach to identify allosteric sites of multimeric enzymes in general.

Electron microscopic evidence for the myosin head lever arm mechanism in hydrated myosin filaments using the gas environmental chamber

Muscle contraction results from an attachment–detachment cycle between the myosin heads extending from myosin filaments and the sites on actin filaments. The myosin head first attaches to actin together with the products of ATP hydrolysis, performs a power stroke associated with release of hydrolysis products, and detaches from actin upon binding with new ATP. The detached myosin head then hydrolyses ATP, and performs a recovery stroke to restore its initial position. The strokes have been suggested to result from rotation of the lever arm domain around the converter domain, while the catalytic domain remains rigid. To ascertain the validity of the lever arm hypothesis in muscle, we recorded ATP-induced movement at different regions within individual myosin heads in hydrated myosin filaments, using the gas environmental chamber attached to the electron microscope. The myosin head were position-marked with gold particles using three different site-directed antibodies. The amplitude of ATP-induced movement at the actin binding site in the catalytic domain was similar to that at the boundary between the catalytic and converter domains, but was definitely larger than that at the regulatory light chain in the lever arm domain. These results are consistent with the myosin head lever arm mechanism in muscle contraction if some assumptions are made.

A kinetic model that explains the effect of inorganic phosphate on the mechanics and energetics of isometric contraction of fast skeletal muscle.

A conventional five-step chemo-mechanical cycle of the myosin-actin ATPase reaction, which implies myosin detachment from actin upon release of hydrolysis products (ADP and phosphate, Pi) and binding of a new ATP molecule, is able to fit the [Pi] dependence of the force and number of myosin motors during isometric contraction of skeletal muscle. However, this scheme is not able to explain why the isometric ATPase rate of fast skeletal muscle is decreased by an increase in [Pi] much less than the number of motors. The question can be solved assuming the presence of a branch in the cycle: in isometric contraction, when the force generation process by the myosin motor is biased at the start of the working stroke, the motor can detach at an early stage of the ATPase cycle, with Pi still bound to its catalytic site, and then rapidly release the hydrolysis products and bind another ATP. In this way, the model predicts that in fast skeletal muscle the energetic cost of isometric contraction increases with [Pi]. The large dissociation constant of the product release in the branched pathway allows the isometric myosin-actin reaction to fit the equilibrium constant of the ATPase.

Substrate interactions and promiscuity in a viral DNA packaging motor

The ASCE superfamily of proteins consists of structurally similar ATPases associated with diverse cellular activities involving metabolism and transport of proteins and nucleic acids in all forms of life1. A subset of these enzymes are multimeric ringed pumps responsible for DNA transport in processes including genome packaging in adenoviruses, herpesviruses, poxviruses, and tailed bacteriophages2. While their mechanism of mechanochemical conversion is beginning to be understood3, little is known about how these motors engage their nucleic acid substrates. Do motors contact a single DNA element, such as a phosphate or a base, or are contacts distributed over multiple parts of the DNA? In addition, what role do these contacts play in the mechanochemical cycle? Here we use the genome packaging motor of the Bacillus subtilis bacteriophage φ294 to address these questions. The full mechanochemical cycle of the motor, whose ATPase is a pentameric-ring5 of gene product 16, involves two phases-- an ATP loading dwell followed by a translocation burst of four 2.5-bp steps6 triggered by hydrolysis product release7. By challenging the motor with a variety of modified DNA substrates, we find that during the dwell phase important contacts are made with adjacent phosphates every 10-bp on the 5’-3’ strand in the direction of packaging. In addition to providing stable, long-lived contacts, these phosphate interactions also regulate the chemical cycle. In contrast, during the burst phase, we find that DNA translocation is driven against large forces by extensive contacts, some of which are not specific to the chemical moieties of DNA. Such promiscuous, non-specific contacts may reflect common translocase-substrate interactions for both the nucleic acid and protein translocases of the ASCE superfamily1.

Measurement Of ATPase Activity During Ramped Stretches In Contracting Skeletal Muscle Fibers Of The Rabbit

Skeletal muscle force response to small amplitude and low velocity ramp stretches is biphasic. An initial fast increase in force, where myosin heads are forcibly detached is followed by a slower increase in force with net re-attachment of myosin heads to actin. The average ATPase rate is very low during stretch (Curtin & Davies,1973), but due to lack of time resolution the two phases to match force changes have never been resolved. We therefore examined and modeled tension and ATPase responses to ramp stretches (5% and 1% of fiber length, Lo) at low velocities (0.1 and 0.5 Lo/s) in permeabilised fiber bundles of rabbit psoas at 12 and 20°C. We show that ATPase activity drops to near zero during the initial fast phase of the stretch and increases slightly but still remains lower than isometric during the second part of the stretch phase, returning to normal post-stretch. The response was not as marked at 12 as at 20°C, although ATPase rate was still reduced in both the fast initial and slow secondary phase of the force response. During the initial phase the myosin heads are forcibly removed from actin, the cross-bridge cycle is not complete and release of hydrolysis products is interrupted. In the second phase, myosin heads re-attach whilst the muscle is still lengthened and the cross-bridge cycle is truncated for a fraction of attached heads, leading to slower Pi release than during isometric conditions. These effects are less marked at 12 than at 20°C because the fraction of strongly bound cross-bridges is reduced at the lower temperature.Curtin, N.A. & Davies, R.E. (1973). Cold Spring Harbor Symp. on Q. Biol., 37, 619-626.

Changes in Mg2+ Ion Concentration and Heavy Chain Phosphorylation Regulate the Motor Activity of a Class I Myosin

Class I myosins are single-headed motor proteins implicated in various motile processes including organelle translocation, ion channel gating, and cytoskeleton reorganization. Dictyostelium discoideum myosin-ID belongs to subclass 1alpha, whose members are thought to be tuned for rapid sliding. The direct analysis of myosin-ID motor activity is made possible by the production of single polypeptide constructs carrying an artificial lever arm. Using these constructs, we show that the motor activity of myosin-ID is activated 80-fold by phosphorylation at the TEDS site. TEDS site phosphorylation acts by stabilizing the actomyosin complex and increasing the coupling between actin binding and the release of hydrolysis products. A surprising effect of Mg(2+) ions on in vitro motility was discovered. Changes in the level of free Mg(2+) ions within the physiological range are shown to modulate motor activity by inhibiting ADP release. Our results indicate that higher concentrations of free Mg(2+) ions stabilize the tension-bearing actin myosin ADP state and shift the system from the production of rapid movement toward the generation of tension.

Regulation of contraction in striated muscle.

Ca(2+) regulation of contraction in vertebrate striated muscle is exerted primarily through effects on the thin filament, which regulate strong cross-bridge binding to actin. Structural and biochemical studies suggest that the position of tropomyosin (Tm) and troponin (Tn) on the thin filament determines the interaction of myosin with the binding sites on actin. These binding sites can be characterized as blocked (unable to bind to cross bridges), closed (able to weakly bind cross bridges), or open (able to bind cross bridges so that they subsequently isomerize to become strongly bound and release ATP hydrolysis products). Flexibility of the Tm may allow variability in actin (A) affinity for myosin along the thin filament other than through a single 7 actin:1 tropomyosin:1 troponin (A(7)TmTn) regulatory unit. Tm position on the actin filament is regulated by the occupancy of NH-terminal Ca(2+) binding sites on TnC, conformational changes resulting from Ca(2+) binding, and changes in the interactions among Tn, Tm, and actin and as well as by strong S1 binding to actin. Ca(2+) binding to TnC enhances TnC-TnI interaction, weakens TnI attachment to its binding sites on 1-2 actins of the regulatory unit, increases Tm movement over the actin surface, and exposes myosin-binding sites on actin previously blocked by Tm. Adjacent Tm are coupled in their overlap regions where Tm movement is also controlled by interactions with TnT. TnT also interacts with TnC-TnI in a Ca(2+)-dependent manner. All these interactions may vary with the different protein isoforms. The movement of Tm over the actin surface increases the "open" probability of myosin binding sites on actins so that some are in the open configuration available for myosin binding and cross-bridge isomerization to strong binding, force-producing states. In skeletal muscle, strong binding of cycling cross bridges promotes additional Tm movement. This movement effectively stabilizes Tm in the open position and allows cooperative activation of additional actins in that and possibly neighboring A(7)TmTn regulatory units. The structural and biochemical findings support the physiological observations of steady-state and transient mechanical behavior. Physiological studies suggest the following. 1) Ca(2+) binding to Tn/Tm exposes sites on actin to which myosin can bind. 2) Ca(2+) regulates the strong binding of M.ADP.P(i) to actin, which precedes the production of force (and/or shortening) and release of hydrolysis products. 3) The initial rate of force development depends mostly on the extent of Ca(2+) activation of the thin filament and myosin kinetic properties but depends little on the initial force level. 4) A small number of strongly attached cross bridges within an A(7)TmTn regulatory unit can activate the actins in one unit and perhaps those in neighboring units. This results in additional myosin binding and isomerization to strongly bound states and force production. 5) The rates of the product release steps per se (as indicated by the unloaded shortening velocity) early in shortening are largely independent of the extent of thin filament activation ([Ca(2+)]) beyond a given baseline level. However, with a greater extent of shortening, the rates depend on the activation level. 6) The cooperativity between neighboring regulatory units contributes to the activation by strong cross bridges of steady-state force but does not affect the rate of force development. 7) Strongly attached, cycling cross bridges can delay relaxation in skeletal muscle in a cooperative manner. 8) Strongly attached and cycling cross bridges can enhance Ca(2+) binding to cardiac TnC, but influence skeletal TnC to a lesser extent. 9) Different Tn subunit isoforms can modulate the cross-bridge detachment rate as shown by studies with mutant regulatory proteins in myotubes and in in vitro motility assays. (ABSTRACT TRUNCATED)

Hydrolysis and Erosion Studies of Autocatalyzed Poly(ortho esters) Containing Lactoyl−Lactyl Acid Dimers

Poly(ortho esters) based on 3,9-diethylidene-2,4,8,10-tetraoxaspiro[5.5]undecane, 1,10-decanediol, and 1,10-decanediol dilactate were prepared and their hydrolysis in phosphate buffer at 37 °C and pH 7.4 was determined as a function of time. Specifically, kinetics of polymer weight loss, polymer molecular weight changes, release of lactic acid and release of propionic acid were determined. A significant decrease in molecular weight occurred within about 20 days. Weight loss and evolution of hydrolysis product occurred after an induction period that decreased with increasing content of the lactoyl-lactyl segment in the polymer. After the induction period, a concomitant rate of weight loss and release of hydrolysis products was noted. The rate increased with increasing content of the lactoyl-lactide segment. After a certain time period, size-exclusion chromatography revealed the formation of peak at an apparent molecular mass of about 550 Da. The compounds under the peak were analyzed by electrospray ionization/ mass spectrometry (ESI/MS) and found to consist of a multiplicity of lower molecular weight polymer hydrolysis fragments. On the basis of expected hydrolysis products, it was possible to assign a structure to most molecular weight peaks in the ESI/MS spectrum. The data were found to be consistent with a hydrolysis that takes place predominantly in the surface layers of the solid polymer.

Evaluation of catalytic activity and synergism between two xylanase isoenzymes in enzymic hydrolysis of two separate xylans in different states of solubility

Synergism of two xylanase (1,4) β- d-xylan xylanohydrolase, EC 3.2.1.8) isoenzymes from Streptomyces A451 was observed in enzymic hydrolysis of water-soluble and -insoluble fractions of two commercial xylan preparations. The reaction modes were determined qualitatively by TLC and quantitatively by GLC. With oat-spelt xylan as substrate, the total amount of xylose and xylobiose produced by using xylanase I and xylanase II in sequence was significantly higher than that by xylanase I or xylanase II individually, while the amounts of the relatively higher-molecular-weight oligosaccharides xylotriose to xylopentaose obtained by using isoxylanases individually were more than those obtained by using isoxylanases in sequence. A time-course study on the release of hydrolysis products over a 24-h period revealed that higher oligosaccharides were released initially followed by release of simpler sugars. A study of the hydrolysis of model oligosaccharides by crude enzyme preparation revealed a preference for higher oligosaccharides and demonstrated that xylopentaose was hydrolyzed to xylose, xylobiose, and xylotriose. A comparison was made of the hydrolysis of two different xylans, birchwood (hardwood) and oat-spelt (softwood), by crude xylanase. Product profiles showed that there were significant differences between the range and amounts of products released from the two different xylans. Studies on relative specific activities showed that xylanase II had overall lower specific activity toward various xylans than did xylanase I, and xylanase I, xylanase II, and crude xylanase showed lower specific activity toward insoluble xylan fractions than toward soluble xylan fractions. In addition, the reduction in specific activity was greater for insoluble birchwood than for insoluble oat-spelt xylan.

Product Release is Not the Rate-Limiting Step during Cytochalasin B-Induced ATPase Activity of Monomeric Actin

Under conditions where cytochalasin B induces ATPase activity of monomeric actin (0.3 mM MgCl2, 1 mM EGTA, 30 microns cytochalasin B, 1 mM ATP) the rate constant of the exchange of actin-bound epsilon-ATP for free ATP is about 4-6 times faster than steady state ATPase activity. When a stoichiometric ATP-actin complex is extracted with PCA (single turnover experiment) the apparent rate constant of Pi generation is not faster than steady state ATPase activity. - The experiments suggest that the hydrolysis of actin-bound ATP and not the subsequent release of hydrolysis products is rate-limiting during cytochalasin-induced ATPase activity of actin.

Induced autolysis ofSaccharomyces cerevisiae: Morphological effects, rheological effects, and dynamics of accumulation of extracellular hydrolysis products

Changes in the internal structure ofSaccharomyces cerevisiae cells and accumulation of proteins and nucleic components in extracellular fluid as decomposition products were studied in the 40–75°C temperature range under the effect of various membranotrophic additives. Autolysis was shown to be a two-step process: The first step consists of the restructuring of cell endostructures and the activation of lytic enzymes, which is accompanied by reduction of cell volume and system viscosity; the second step directly follows the first step and consists of hydrolysis of cell components and release of hydrolysis products into extracellular space. Duration of the first step depends on the temperature and the plasmolyzer. Hydrophilic additives (ethanol, ethyl acetate) were most effective during the first step at 60–65°C, whereas hydrophobic additives (lecithin, lauric acid) were most effective at 55°C. In the second step, the temperature optimum of protease activity in the control (without additives) was 60°C, that of nuclease activity was 70°C. Additives reduce the temperature optimum of endoenzymatic activity. Cell morphology was studied at various stages of autolysis by electron and phase-contrast microscopy.