Pi Release Research Articles

A human skeletal muscle cross‐bridge model to characterize the role of metabolite accumulation in muscle fatigue

Abstract Skeletal muscle fatigue is accompanied by the accumulation of metabolites, such as adenosine diphosphate (ADP), inorganic phosphate (Pi), and protons (H+). However, we lack a comprehensive understanding of the contribution of these metabolic changes to the development of muscle fatigue during intense exercise and the underlying mechanisms. To address this gap, we collected data from young adults performing a dynamic (0.75 Hz) plantar flexion exercise to task failure (642 ± 104 s), including in vivo concentrations of metabolites and H+ measured by 31P magnetic resonance spectroscopy as well as muscle activation signals obtained via electromyography. Using these data, we developed and validated a human skeletal muscle model. Our model‐based simulations suggested that to continue the plantar flexion exercise at the required power output, muscle activation should progressively increase. In the absence of this increased activation, we observed a reduction in force‐generating capacity due to metabolite‐mediated inhibition of actin–myosin cross‐bridge cycling. Our simulations also showed that Pi reduced force production by 30% when we increased it 50% above the concentrations measured experimentally. A parameter sensitivity analysis suggested that force generation is strongly dependent on the rate of Pi release from the actin–myosin complex, and Pi inhibits force by increasing the rate of actin–myosin detachment. In addition, we proposed an alternative mechanism through which H+ might reduce muscle force generation during exercise. In contrast, elevated ADP levels did not significantly affect force generation. This study provides insight into the impact of metabolite accumulation on force generation and muscle fatigue development.

A Soluble Expression Construct of the Isolated Catalytic Domain of Plasmodium falciparumATP4 Exhibits ATPase Activity Independent of a γ‐Phosphate Receiving Aspartate

ABSTRACTThe sodium/proton‐exchanging ATPase of Plasmodium falciparum malaria parasites, PfATP4, is an emerging drug target. Inhibition results in detrimental cell swelling due to cytosolic accumulation of sodium and alkalization. PfATP4 is a sodium‐releasing type II P‐type ATPase restricted to apicomplexan parasites. Experimental data on structure–function relationships of the isolated protein are absent. Here, we produced and purified the soluble catalytic domain of PfATP4 and evaluated kinetic properties by in vitro phosphate colorimetry. The protein exhibited Mg2+‐dependent ATPase activity at the same order of magnitude as the native cellular PfATP4 and was insensitive to the presence of sodium. AlphaFold 3‐based structure and ATP/Mg2+ interaction predictions identified key residues of the nucleotide binding domain (Lys619, Lys652, Arg703). Replacement of the lysines by methionine decreased the enzymatic activity to one quarter. Individual mutation of the putative Mg2+‐coordinating Asp865 of the phosphorylation domain was tolerated, while a joint replacement with Asp869 decreased ATPase again to one quarter. Mutation of the putative γ‐phosphate receiving Asp451 maintained the rate of Pi release. Our data attribute typical functional roles for P‐type ATPases to the basic and acidic residues of the soluble PfATP4 catalytic domain and show that its ATP hydrolysis is independent of phosphorylation of Asp451.

Catalytic dwell oscillations complete the F1-ATPase mechanism

The F1-ATPase molecular motor rotates subunit-γ in 120° power strokes within its ring of three catalytic sites separated by catalytic dwells for ATP hydrolysis and Pi release. By monitoring rotary position of subunit-γ in E. coli F1 every 5 μs, we resolved Stage-1 catalytic dwell oscillations that extend from -13° to 13° centered at 0° consistent with F1 structures containing transition state inhibitors, which decay by a first order process consistent with ATP hydrolysis. During Stage-2, 80% of the oscillations extend from 3° and 25° centered at 14°, while 20% are centered at 33° and can extend to 27°–44° comparable to the ATP binding position. Remarkably, in Stage-3 subunit-γ returns to 0° to end the catalytic dwell, which keeps the start of power strokes in phase for consecutive rotational events. These newly observed states fit with F1 structures that were inconsistent with the canonical mechanism, and indicate that catalytic dwell oscillations must persist until the correct occupancy of substrates and products occurs at all three catalytic sites. When that condition is met, F1 can proceed to the next power stroke. Understanding the basis of these catalytic dwell oscillations completes the F1-ATPase rotary mechanism.

A Phosphate-Starvation Enhanced Purple Acid Phosphatase, GmPAP23 Mediates Intracellular Phosphorus Recycling and Yield in Soybean.

Plant internal phosphorus (P) recycling is a complex process, which is vital for improving plant P use efficiency. However, the mechanisms underlying phosphate (Pi) release from internal organic-P form remains to be deciphered in crops. Here, we functionally characterised a Pi-starvation responsive purple acid phosphatase (PAP), GmPAP23 in soybean (Glycine max). GmPAP23 could hydrolyse a series of Pi-containing compounds in vitro, such as trehalose-6-phosphate and glucose-l-phosphate. Moreover, GmPAP23 overexpression led to less P distribution in soybean source organs, including mature leaves and pod shells, but more P distribution in seeds under P sufficient conditions, although no effect was observed for transgenic soybean lines with its suppression. Metabolomic analysis found that a group of P-containing metabolites exhibited differential accumulations in mature leaves between wild type (WT) and GmPAP23 overexpression lines, such as glucose-l-phosphate and trehalose-6-phosphate. Moreover, a MYB transcription factor, GmPHR14 was subsequently found to activate the transcription of GmPAP23 via directly binding to its promoter. Collectively, these findings could highlight that the GmPHR14-GmPAP23 pathway, which controls internal P recycling in soybean, and thus affect yield.

Phosphate rebinding induces force reversal via slow backward cycling of cross-bridges.

Previous studies on muscle fibers, myofibrils, and myosin revealed that the release of inorganic phosphate (Pi) and the force-generating step(s) are reversible, with cross-bridges also cycling backward through these steps by reversing force-generating steps and rebinding Pi. The aim was to explore the significance of force redevelopment kinetics (rate constant k TR) in cardiac myofibrils for the coupling between the Pi binding induced force reversal and the rate-limiting transition f - for backward cycling of cross-bridges from force-generating to non-force-generating states. k TR and force generation of cardiac myofibrils from guinea pigs were investigated at 0.015-20mM Pi. The observed force-[Pi], force-log [Pi], k TR-[Pi], and k TR-force relations were assessed with various single-pathway models of the cross-bridge cycle that differed in sequence and kinetics of reversible Pi release, reversible force-generating step and reversible rate-limiting transition. Based on the interpretation that k TR reflects the sum of rate-limiting transitions in the cross-bridge cycle, an indicator, the coupling strength, was defined to quantify the contribution of Pi binding induced force reversal to the rate-limiting transition f - from the [Pi]-modulated k TR-force relation. Increasing [Pi] decreased force by a bi-linear force-log [Pi] relation, increased k TR in a slightly downward curved dependence with [Pi], and altered k TR almost reciprocally to force reflected by the k TR-force relation. Force-[Pi] and force-log [Pi] relations provided less selectivity for the exclusion of models than the k TR-[Pi] and k TR-force relations. The k TR-force relation observed in experiments with cardiac myofibrils yielded the coupling strength +0.84 ± 0.08 close to 1, the maximum coupling strength expected for the reciprocal k TR-force relationship. Single pathway models consisting of fast reversible force generation before or after rapid reversible Pi release failed to describe the observed k TR-force relation. Single pathway models consistent with the observed k TR-force relation had either slow Pi binding or slow force reversal, i.e., in the consistent single pathway models, f - was assigned to the rate of either Pi binding or force reversal. Backward flux of cross-bridges from force-generating to non-force-generating states is limited by the rates of Pi binding or force reversal ruling out other rate-limiting steps uncoupled from Pi binding induced force reversal.

Osteoclast formation in the co-presence of octacalcium phosphate and inorganic phosphorus-containing gelatin in vitro.

This study was designed to investigate the effect of biodegradable octacalcium phosphate (OCP) bone substitute on osteoclast formation under the higher inorganic phosphorus concentration in vitro. We developed the gelatin particles releasing inorganic phosphate (Pi) ions as a model to change the local Pi ion concentration around OCP in the OCP/gelatin composite materials. Spectroscopic and chemical analyses indicated that Pi ion released around 2 mmol/L from gelatin particles promoted OCP hydrolysis, although excessive Pi ion release up to around 3 mmol/L suppressed the hydrolysis. The formation of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells was increased by the OCP hydrolysis promotion via modulately higher Pi release Pi ion release. However, OCP hydrolysis suppression by excessive Pi ion release decreased the formation of TRAP-positive cells. The present study suggests that the chemical environment induced by OCP hydrolysis under a suitable Pi ion concentration could be associated with promoting osteoclast formation.

Single-Molecule Investigation of Load-Dependent Actomyosin Dissociation Kinetics for Cardiac and Slow Skeletal Myosin.

Myosins are ATP-powered, force-generating motor proteins involved in cardiac and muscle contraction. The external load experienced by the myosins modulates and coordinates their function in vivo. Here, this study investigates the tension-sensing mechanisms of rabbit native β-cardiac myosin (βM-II) and slow skeletal myosins (SolM-II) that perform in different physiological settings. Using mobile optical tweezers with a square wave-scanning mode, a range of external assisting and resisting loads from 0 to 15 pN is exerted on single myosin molecules as they interact with the actin filament. Influenced of load on specific strongly-bound states in the cross-bridge cycle is examined by adjusting the [ATP]. The results implies that the detachment kinetics of actomyosin ADP.Pi strongly-bound force-generating state are load sensitive. Low assisting load accelerates, while the resisting load hinders the actomyosin detachment, presumably, by slowing both the Pi and ADP release. However, under both high assisting and resisting load, the rate of actomyosin dissociation decelerates. The transition from actomyosin ADP.Pi to ADP state appears to occur with a higher probability for βM-II than SolM-II. This study interpret that dissociation of at least three strongly-bound actomyosin states are load-sensitive and may contribute to functional diversity among different myosins.

Myosin Isoform-Dependent Effect of Omecamtiv Mecarbil on the Regulation of Force Generation in Human Cardiac Muscle.

Omecamtiv mecarbil (OM) is a small molecule that has been shown to improve the function of the slow human ventricular myosin (MyHC) motor through a complex perturbation of the thin/thick filament regulatory state of the sarcomere mediated by binding to myosin allosteric sites coupled to inorganic phosphate (Pi) release. Here, myofibrils from samples of human left ventricle (β-slow MyHC-7) and left atrium (α-fast MyHC-6) from healthy donors were used to study the differential effects of μmolar [OM] on isometric force in relaxing conditions (pCa 9.0) and at maximal (pCa 4.5) or half-maximal (pCa 5.75) calcium activation, both under control conditions (15 °C; equimolar DMSO; contaminant inorganic phosphate [Pi] ~170 μM) and in the presence of 5 mM [Pi]. The activation state and OM concentration within the contractile lattice were rapidly altered by fast solution switching, demonstrating that the effect of OM was rapid and fully reversible with dose-dependent and myosin isoform-dependent features. In MyHC-7 ventricular myofibrils, OM increased submaximal and maximal Ca2+-activated isometric force with a complex dose-dependent effect peaking (40% increase) at 0.5 μM, whereas in MyHC-6 atrial myofibrils, it had no effect or-at concentrations above 5 µM-decreased the maximum Ca2+-activated force. In both ventricular and atrial myofibrils, OM strongly depressed the kinetics of force development and relaxation up to 90% at 10 μM [OM] and reduced the inhibition of force by inorganic phosphate. Interestingly, in the ventricle, but not in the atrium, OM induced a large dose-dependent Ca2+-independent force development and an increase in basal ATPase that were abolished by the presence of millimolar inorganic phosphate, consistent with the hypothesis that the widely reported Ca2+-sensitising effect of OM may be coupled to a change in the state of the thick filaments that resembles the on-off regulation of thin filaments by Ca2+. The complexity of this scenario may help to understand the disappointing results of clinical trials testing OM as inotropic support in systolic heart failure compared with currently available inotropic drugs that alter the calcium signalling cascade.

Structural transitions in kinesin minus-end directed microtubule motility

Kinesin motor proteins hydrolyze ATP to produce force for spindle assembly and vesicle transport, performing essential functions in cell division and motility, but the structural changes required for force generation are uncertain. We now report high-resolution structures showing new transitions in the kinesin mechanochemical cycle, including power stroke fluctuations upon ATP binding and a post-hydrolysis state with bound ADP + free phosphate. We find that rate-limiting ADP release occurs upon microtubule binding, accompanied by central β-sheet twisting, which triggers the power stroke – stalk rotation and neck mimic docking – upon ATP binding. Microtubule release occurs with β-strand-to-loop transitions, implying that β-strand refolding induces Pi release and the recovery stroke. The strained β-sheet during the power stroke and strand-to-loop transitions identify the β-sheet as the long-sought motor spring.

Human cardiac β-myosin powerstroke energetics: Thin filament, Pi displacement, and mutation effects

Human cardiac β-myosin powerstroke energetics: Thin filament, Pi displacement, and mutation effects

Mechanism of phosphate release from actin filaments

After ATP-actin monomers assemble filaments, the ATP's [Formula: see text]-phosphate is hydrolyzedwithin seconds and dissociates over minutes. We used all-atom molecular dynamics simulations to sample the release of phosphate from filaments and study residues that gate release. Dissociation of phosphate from Mg2+ is rate limiting and associated with an energy barrier of 20 kcal/mol, consistent with experimental rates of phosphate release. Phosphate then diffuses within an internal cavity toward a gate formed by R177, as suggested in prior computational studies and cryo-EM structures. The gate is closed when R177 hydrogen bonds with N111 and is open when R177 forms a salt bridge with D179. Most of the time, interactions of R177 with other residues occlude the phosphate release pathway. Machine learning analysis reveals that the occluding interactions fluctuate rapidly, underscoring the secondary role of backdoor gate opening in Pi release, in contrast with the previous hypothesis that gate opening is the primary event.

Multicomponent depolymerization of actin filament pointed ends by cofilin and cyclase-associated protein depends upon filament age

Intracellular actin networks assemble through the addition of ATP-actin subunits at the growing barbed ends of actin filaments. This is followed by “aging” of the filament via ATP hydrolysis and subsequent phosphate release. Aged ADP-actin subunits thus “treadmill” through the filament before being released back into the cytoplasmic monomer pool as a result of depolymerization at filament pointed ends. The necessity for aging before filament disassembly is reinforced by preferential binding of cofilin to aged ADP-actin subunits over newly-assembled ADP-Pi actin subunits in the filament. Consequently, investigations into how cofilin influences pointed-end depolymerization have, thus far, focused exclusively on aged ADP-actin filaments. Using microfluidics-assisted Total Internal Reflection Fluorescence (mf-TIRF) microscopy, we reveal that, similar to their effects on ADP filaments, cofilin and cyclase-associated protein (CAP) also promote pointed-end depolymerization of ADP-Pi filaments. Interestingly, the maximal rates of ADP-Pi filament depolymerization by CAP and cofilin together remain approximately 20–40 times lower than for ADP filaments. Further, we find that the promotion of ADP-Pi pointed-end depolymerization is conserved for all three mammalian cofilin isoforms. Taken together, the mechanisms presented here open the possibility of newly-assembled actin filaments being directly disassembled from their pointed-ends, thus bypassing the slow step of Pi release in the aging process.

Oscillatory work and the step that generates force in single myofibrils from rabbit psoas.

The elementary molecular step that generates force by cross-bridges (CBs) in active muscles has been under intense investigation in the field of muscle biophysics. It is known that an increase in the phosphate (Pi) concentration diminishes isometric force in active fibers, indicating a tight coupling between the force generation step and the Pi release step. The question asked here is whether the force generation occurs before Pi release or after release. We investigated the effect of Pi on oscillatory work production in single myofibrils and found that Pi-attached state(s) to CBs is essential for its production. Oscillatory work is the mechanism that allows an insect to fly by beating its wings, and it also has been observed in skeletal and cardiac muscle fibers, implying that it is an essential feature of all striated muscle types. With our studies, oscillatory work disappears in the absence of Pi in experiments using myofibrils. This suggests that force is generated during a transition between steps of oscillatory work production, and that the states involved in force production must have Pi attached. With sinusoidal analysis, we obtained the kinetic constants around the Pi release steps, established a CB scheme, and evaluated force generated (and supported) by each CB state. Our results demonstrate that force is generated before Pi is released, and the same force is maintained after Pi is released. Stretch activation and/or delayed tension can also be explained with this CB scheme and forms the basis of force generation and oscillatory work production.

An association analysis of lipidome and transcriptome highlights the involvement of MmGDPD1 in regulating low phosphorus tolerance in Malus mandshurica

An association analysis of lipidome and transcriptome highlights the involvement of MmGDPD1 in regulating low phosphorus tolerance in Malus mandshurica

Dissecting the mechanism of atlastin-mediated homotypic membrane fusion at the single-molecule level

Homotypic membrane fusion of the endoplasmic reticulum (ER) is mediated by dynamin-like GTPase atlastin (ATL). This fundamental process relies on GTP-dependent domain rearrangements in the N-terminal region of ATL (ATLcyto), including the GTPase domain and three-helix bundle (3HB). However, its conformational dynamics during the GTPase cycle remain elusive. Here, we combine single-molecule FRET imaging and molecular dynamics simulations to address this conundrum. Different from the prevailing model, ATLcyto can form a loose crossover dimer upon GTP binding, which is tightened by GTP hydrolysis for membrane fusion. Furthermore, the α-helical motif between the 3HB and transmembrane domain, which is embedded in the surface of the lipid bilayer and self-associates in the crossover dimer, is required for ATL function. To recycle the proteins, Pi release, which disassembles the dimer, activates frequent relative movements between the GTPase domain and 3HB, and subsequent GDP dissociation alters the conformational preference of the ATLcyto monomer for entering the next reaction cycle. Finally, we found that two disease-causing mutations affect human ATL1 activity by destabilizing GTP binding-induced loose crossover dimer formation and the membrane-embedded helix, respectively. These results provide insights into ATL-mediated homotypic membrane fusion and the pathological mechanisms of related disease.

Steric blocking upside down: a different way of thinking about the competition between myosin and tropomyosin

At low free Ca2+, the actin binding proteins tropomyosin, troponin I, troponin T and troponin C inhibit contraction in striated muscles. Ca2+ activation alters the position of tropomyosin on actin to uncover binding sites for high affinity forms of myosin (i.e., myosin-ADP). Inhibition of contraction is commonly thought to result from steric blocking of myosin binding to actin by tropomyosin. However, myosin-ADP binding to actin is energetically more favorable than localization of tropomyosin in the blocking position. Tropomyosin is an effective inhibitor of binding only at low levels of myosin-ADP. At low free Ca2+, troponin-tropomyosin also inhibits the rate of a step associated with Pi release to about 1% of the maximum rate. This results in accumulation of myosin with bound ATP and ADP-Pi. Such myosin binds weakly to actin. Ca2+ activation increases the rate of Pi release, but not to the maximum value, and increases the population of myosin-ADP. The high affinity binding of myosin-ADP to actin can displace tropomyosin into the fully active position in relation to the amount of myosin-ADP bound. It seems likely that an important outcome of the steric clash between myosin-ADP and tropomyosin is the dual activation by Ca2+ and myosin-ADP. The C-terminal region of troponin T (TnT) contributes to the incomplete activation by Ca2+ alone. Because this region of TnT is highly conserved, the ability of myosin-ADP to move tropomyosin may be more important than any restriction that tropomyosin may place on myosin binding.

Analysis of metabolite and strain effects on cardiac cross-bridge dynamics using model linearisation techniques.

Multi-scale models of cardiac energetics are becoming crucial in better understanding the prevalent chronic diseases operating at the intersection of metabolic and cardiovascular dysfunction. Computationally efficient models of cardiac cross-bridge kinetics that are sensitive to changes in metabolite concentrations are necessary to simulate the effects of disease-induced changes in cellular metabolic state on cardiac mechanics across disparate spatial scales. While these models do currently exist, deeper analysis of how the modelling of metabolite effects and the assignment of strain dependence within the cross-bridge cycle affect the properties of the model is required. In this study, model linearisation techniques were used to simulate and interrogate the complex modulus of an ODE-based model of cross-bridge kinetics. Active complex moduli were measured from permeabilised rat cardiac trabeculae under five different metabolite conditions with varying ATP and Pi concentrations. Sensitivity to metabolites was incorporated into an existing three-state cross-bridge model using either a direct dependence or a rapid equilibrium approach. Combining the two metabolite binding methods with all possible locations of strain dependence within the cross-bridge cycle produced 64 permutations of the cross-bridge model. Using linear model analysis, these models were systematically explored to determine the effects of metabolite binding and their interaction with strain dependence on the frequency response of cardiac muscle. The results showed that the experimentally observed effects of ATP and Pi concentrations on the cardiac complex modulus could be attributed to their regulation of cross-bridge detachment rates. Analysis of the cross-bridge models revealed a mechanistic basis for the biochemical schemes which place Pi release following cross-bridge formation and ATP binding prior to cross-bridge detachment. In addition, placing strain dependence on the reverse rate of the cross-bridge power stroke produced the model which most closely matched the experimental data. From these analyses, a well-justified metabolite-sensitive model of rat cardiac cross-bridge kinetics is presented which is suitable for parameterisation with other data sets and integration with multi-scale cardiac models.

Molecular mechanisms of inorganic-phosphate release from the core and barbed end of actin filaments.

The release of inorganic phosphate (Pi) from actin filaments constitutes a key step in their regulated turnover, which is fundamental to many cellular functions. The mechanisms underlying Pi release from the core and barbed end of actin filaments remain unclear. Here, using human and bovine actin isoforms, we combine cryo-EM with molecular-dynamics simulations and in vitro reconstitution to demonstrate how actin releases Pi through a 'molecular backdoor'. While constantly open at the barbed end, the backdoor is predominantly closed in filament-core subunits and opens only transiently through concerted amino acid rearrangements. This explains why Pi escapes rapidly from the filament end but slowly from internal subunits. In a nemaline-myopathy-associated actin variant, the backdoor is predominantly open in filament-core subunits, resulting in accelerated Pi release and filaments with drastically shortened ADP-Pi caps. Our results provide the molecular basis for Pi release from actin and exemplify how a disease-linked mutation distorts the nucleotide-state distribution and atomic structure of the filament.

Beyond binding change: the molecular mechanism of ATP hydrolysis by F1-ATPase and its biochemical consequences

F1-ATPase is a universal multisubunit enzyme and the smallest-known motor that, fueled by the process of ATP hydrolysis, rotates in 120o steps. A central question is how the elementary chemical steps occurring in the three catalytic sites are coupled to the mechanical rotation. Here, we performed cold chase promotion experiments and measured the rates and extents of hydrolysis of preloaded bound ATP and promoter ATP bound in the catalytic sites. We found that rotation was caused by the electrostatic free energy change associated with the ATP cleavage reaction followed by Pi release. The combination of these two processes occurs sequentially in two different catalytic sites on the enzyme, thereby driving the two rotational sub-steps of the 120o rotation. The mechanistic implications of this finding are discussed based on the overall energy balance of the system. General principles of free energy transduction are formulated, and their important physical and biochemical consequences are analyzed. In particular, how exactly ATP performs useful external work in biomolecular systems is discussed. A molecular mechanism of steady-state, trisite ATP hydrolysis by F1-ATPase, consistent with physical laws and principles and the consolidated body of available biochemical information, is developed. Taken together with previous results, this mechanism essentially completes the coupling scheme. Discrete snapshots seen in high-resolution X-ray structures are assigned to specific intermediate stages in the 120o hydrolysis cycle, and reasons for the necessity of these conformations are readily understood. The major roles played by the “minor” subunits of ATP synthase in enabling physiological energy coupling and catalysis, first predicted by Nath's torsional mechanism of energy transduction and ATP synthesis 25 years ago, are now revealed with great clarity. The working of nine-stepped (bMF1, hMF1), six-stepped (TF1, EF1), and three-stepped (PdF1) F1 motors and of the α3β3γ subcomplex of F1 is explained by the same unified mechanism without invoking additional assumptions or postulating different mechanochemical coupling schemes. Some novel predictions of the unified theory on the mode of action of F1 inhibitors, such as sodium azide, of great pharmaceutical importance, and on more exotic artificial or hybrid/chimera F1 motors have been made and analyzed mathematically. The detailed ATP hydrolysis cycle for the enzyme as a whole is shown to provide a biochemical basis for a theory of “unisite” and steady-state multisite catalysis by F1-ATPase that had remained elusive for a very long time. The theory is supported by a probability-based calculation of enzyme species distributions and analysis of catalytic site occupancies by Mg-nucleotides and the activity of F1-ATPase. A new concept of energy coupling in ATP synthesis/hydrolysis based on fundamental ligand substitution chemistry has been advanced, which offers a deeper understanding, elucidates enzyme activation and catalysis in a better way, and provides a unified molecular explanation of elementary chemical events occurring at enzyme catalytic sites. As such, these developments take us beyond binding change mechanisms of ATP synthesis/hydrolysis proposed for oxidative phosphorylation and photophosphorylation in bioenergetics.

A mutation in switch I alters the load-dependent kinetics of myosin Va

Myosin Va is the molecular motor that drives intracellular vesicular transport, powered by the transduction of chemical energy from ATP into mechanical work. The coupling of the powerstroke and phosphate (Pi) release is key to understanding the transduction process, and crucial details of this process remain unclear. Therefore, we determined the effect of elevated Pi on the force-generating capacity of a mini-ensemble of myosin Va S1 (WT) in a laser trap assay. By increasing the stiffness of the laser trap we determined the effect of increasing resistive loads on the rate of Pi-induced detachment from actin, and quantified this effect using the Bell approximation. We observed that WT myosin generated higher forces and larger displacements at the higher laser trap stiffnesses in the presence of 30 mM Pi, but binding event lifetimes decreased dramatically, which is most consistent with the powerstroke preceding the release of Pi from the active site. Repeating these experiments using a construct with a mutation in switch I of the active site (S217A) caused a seven-fold increase in the load-dependence of the Pi-induced detachment rate, suggesting that the S217A region of switch I may help mediate the load-dependence of Pi-rebinding.