Free Energy Of Hydrolysis Research Articles

Interplay between ATP and hydrolysis free energy in promoting and restraining biochemical switches

In living cells, the bistable switches involving phosphorylation-dephosphorylation (PdP) cycles, operate far from thermodynamic equilibrium and consume hydrolysis free energy to perform ultrasensitive response and exhibit strong robustness against noise. Here, we focus on the distinct roles of cellular ATP levels ([ATP]) and hydrolysis free energy (Δμ) in bistable switches with different structures of reaction network. Specifically, we herein propose thermodynamically consistent models of single- and double-PdP-cycle bistable switches to study the relationship between network structures and the interplay between [ATP] and Δμ. We demonstrate that [ATP] and Δμ act cooperatively to promote the activation of switches in identical networks wherein all phosphorylations promote target production, irrespective of different feedback mechanisms. On the other hand, the competition between [ATP] and Δμ found in the nonidentical, or reversed, networks can enhance the robustness of bistability. We also discuss special network structures inducing multistability or nonmonotonic functions of Δμ. Our results gain insight into the relationship between network structures and the roles of [ATP] and Δμ in bistable switches, potentially guiding the future design of synthetic biochemical switches. Published by the American Physical Society 2024

ATP hydrolysis kinetics and thermodynamics as determinants of calcium oscillation in pancreatic β cells

Cellular ATP plays an important role in the calcium oscillation signal transduction pathway of pancreatic $\ensuremath{\beta}$ cells. It triggers oscillation by binding the ATP-sensitive ${\text{K}}^{+}$ channels $({\text{K}}_{\mathrm{ATP}})$, and maintains the oscillation by providing ATP hydrolysis free energy for the normal function of ion pumps on the plasma membrane. To reveal how cellular ATP level and ATP hydrolysis free energy affect calcium oscillation, we first constructed a simplified kinetic model of ${\text{K}}_{\mathrm{ATP}}$ and calcium pumps, then analyzed their thermodynamic characteristics. Bifurcation of calcium oscillation is determined by both cellular ATP concentration and ATP hydrolysis free energy such that an insufficient ATP energy supply would result in dysfunctional calcium oscillation. Second, to investigate the glucose sensing in $\ensuremath{\beta}$ cells, we developed a glycolysis-calcium model that considers the dynamics of ATP and free energy levels. The model simulated three calcium patterns in wild type cells and impaired calcium response of ${\text{K}}_{\mathrm{ATP}}$ mutant cells, allowing the use of the ATP-free energy phase plane to explore the underlying mechanism. Our results reveal the thermodynamics of calcium oscillation and provide a framework for understanding the thermodynamics of other ion transport systems.

How Cytoplasmic Dynein Couples ATP Hydrolysis Cycle to Diverse Stepping Motions: Kinetic Modeling

Cytoplasmic dynein is a two-headed molecular motor that moves to the minus end of a microtubule by ATP hydrolysis free energy. By employing its two heads (motor domains), cytoplasmic dynein exhibits various bipedal stepping motions: inchworm and hand-over-hand motions, as well as nonalternating steps of one head. However, the molecular basis to achieve such diverse stepping manners remains unclear because of the lack of an experimental method to observe stepping and the ATPase reaction of dynein simultaneously. Here, we propose a kinetic model for bipedal motions of cytoplasmic dynein and perform Gillespie Monte Carlo simulations that qualitatively reproduce most experimental data obtained to date. The model represents the status of each motor domain as five states according to conformation and nucleotide- and microtubule-binding conditions of the domain. In addition, the relative positions of the two domains were approximated by three discrete states. Accompanied by ATP hydrolysis cycles, the model dynein stochastically and processively moved forward in multiple steps via diverse pathways, including inchworm and hand-over-hand motions, similarly to experimental data. The model reproduced key experimental motility-related properties, including velocity and run length, as functions of the ATP concentration and external force, therefore providing a plausible explanation of how dynein achieves various stepping manners with explicit characterization of nucleotide states. Our model highlights the uniqueness of dynein in the coupling of ATPase with its movement during both inchworm and hand-over-hand stepping.

ZnCl2 Enhanced Acid Hydrolysis of Pretreated Corncob for Glucose Production: Kinetics, Thermodynamics and Optimization Analysis

The biomass of agricultural wastes as a source of fermentable sugars for biofuels production will address the food security and environmental preservation issues. These wastes are rich in lignocellulosic materials which can be hydrolyzed into fermentable sugars. However, low sugar yield and high energy consumption are some of the challenges faced in the process of hydrolization. This study investigated the low-cost corncob substrate for glucose production by dilute sulphuric acid hydrolysis in the presence of ZnCl2 at temperatures below 100°C after pretreatment with 10% NaOH.Time dependent hydrolysis data were analyzed by Saeman model, and thermodynamic parameters were obtained using Erying and Arrhenius equations while Box-Behnken model (Design Expert 6.0 version) was used for experimental design. As the substrate concentration increased from 50 mg/L to 150 mg/L, glucose yield increased from 10.4 mg/g to 14.6 mg/g for pretreated corncob while an increase from 3.4 mg/g to 8.6 mg/g was noted for untreated corncob. The hydrolysis rate constant was two orders of magnitude higher than the degradation rate constant. Thermodynamic parameters revealed endothermic process with positive Gibb’s free energy of hydrolysis having average values of 84.76 kJ/mol and 79.87 kJ/mol for pretreated and untreated samples respectively. The optimum yield from the model was found to be 177.44 mg/g with 3.94% H2SO4 and 0.43 mol/L ZnCl2 for 200 g/L compared with optimum yield of 46.37 mg/g obtainable without ZnCl2. The results of this study showed that the alkaline pretreatment of corncob increased the accessibility of cellulose from the solid fraction and increased glucose production.

Escapement mechanisms: Efficient free energy transduction by reciprocally-coupled gating.

Conversion of the free energy of NTP hydrolysis efficiently into mechanical work and/or information by transducing enzymes sustains living systems far from equilibrium, and so has been of interest for many decades. Detailed molecular mechanisms, however, remain puzzling and incomplete. We previously reported that catalysis of tryptophan activation by tryptophanyl-tRNA synthetase, TrpRS, requires relative domain motion to re-position the catalytic Mg2+ ion, noting the analogy between that conditional hydrolysis of ATP and the escapement mechanism of a mechanical clock. The escapement allows the time-keeping mechanism to advance discretely, one gear at a time, if and only if the pendulum swings, thereby converting energy from the weight driving the pendulum into rotation of the hands. Coupling of catalysis to domain motion, however, mimics only half of the escapement mechanism, suggesting that domain motion may also be reciprocally coupled to catalysis, completing the escapement metaphor. Computational studies of the free energy surface restraining the domain motion later confirmed that reciprocal coupling: the catalytic domain motion is thermodynamically unfavorable unless the PPi product is released from the active site. These two conditional phenomena-demonstrated together only for the TrpRS mechanism-function as reciprocally-coupled gates. As we and others have noted, such an escapement mechanism is essential to the efficient transduction of NTP hydrolysis free energy into other useful forms of mechanical or chemical work and/or information. Some implementation of both gating mechanisms-catalysis by domain motion and domain motion by catalysis-will thus likely be found in many other systems.

Screening for Improved Nerve Agent Simulants and Insights into Organophosphate Hydrolysis Reactions from DFT and QSAR Modeling.

The effective detoxification of chemical warfare agents, specifically nerve agents, is a pressing issue in the modern world. Due to the high toxicity of these molecules, simulants are often used in experiments as substitutes for the agents. However, there is little reason to believe that the current simulants used in the literature are optimal predictors of nerve agent reactivity. Density functional theory calculations were performed on the alkaline hydrolysis of over 100 organophosphate molecules to identify improved simulants for the G-series nerve agents soman and sarin, based on low toxicity and similarity to nerve agent hydrolysis energetics and degradation mechanism. This screening highlighted 5 molecules that have nearly identical reaction barriers to the actual agents, while being far less toxic. Quantitative structure-activity relationship (QSAR) models were also derived to determine the most significant molecular descriptors for describing the hydrolysis free energy barriers of these reactions. The optimal QSAR model was subjected to a thorough statistical analysis and validation procedure to confirm its predictive capacity, showing excellent quantitative and ranking accuracy. It was further shown that the model trained on G-series agents can reliably predict energetics for other organophosphate classes as well, including VX. Through these computational insights, experimentalists may be aided in accurately and safely studying these reactions with less toxic simulants.

Sulfonium Ion Condensation: The Burden Borne by SAM Synthetase

S-Adenosylmethionine (SAM+) serves as the principal methylating agent in biological systems, but the thermodynamic basis of its reactivity does not seem to have been clearly established. Here, we show that methionine, methanol, and H+ combine to form S-methylmethionine (SMM+) with a temperature-independent equilibrium constant of 9.9 M-2. The corresponding group transfer potential of SMM+ (its free energy of hydrolysis at pH 7) is -8.2 kcal/mol. The "energy-rich" nature of sulfonium ions is related to the extreme acidity (p Ka -5.4) of the S-protonated thioether produced by sulfonium hydrolysis, and the large negative free energy of deprotonation of that species in neutral solution (-16.7 kcal/mol). At pH 7, SAM synthetase requires the free energy released by cleavage of two bonds of ATP to reverse that process.

Analytical comparison of natural and pharmaceutical ventricular myosin activators.

Ventricular myosin (βMys) is the motor protein in cardiac muscle generating force using ATP hydrolysis free energy to translate actin. In the cardiac muscle sarcomere, myosin and actin filaments interact cyclically and undergo rapid relative translation facilitated by the low duty cycle motor. It contrasts with high duty cycle processive myosins for which persistent actin association is the priority. The only pharmaceutical βMys activator, omecamtive mecarbil (OM), upregulates cardiac contractility in vivo and is undergoing testing for heart failure therapy. In vitro βMys step-size, motility velocity, and actin-activated myosin ATPase were measured to determine duty cycle in the absence and presence of OM. A new parameter, the relative step-frequency, was introduced and measured to characterize βMys motility due to the involvement of its three unitary step-sizes. Step-size and relative step-frequency were measured using the Qdot assay. OM decreases motility velocity 10-fold without affecting step-size, indicating a large increase in duty cycle converting βMys to a near processive myosin. The OM conversion dramatically increases force and modestly increases power over the native βMys. Contrasting motility modification due to OM with that from the natural myosin activator, specific βMys phosphorylation, provides insight into their respective activation mechanisms and indicates the boilerplate screening characteristics desired for pharmaceutical βMys activators. New analytics introduced here for the fast and efficient Qdot motility assay create a promising method for high-throughput screening of motor proteins and their modulators.

Similarity in Transcytosis of nNOSα in Enteric Nerve Terminals and Beta Cells of Pancreatic Islet.

Transcytosis of proteins and hydrodynamic flow of cytoplasm is a major mechanism to sustain physiology in all cells, observable from gametes (1) to mature adult cells and tissues (2, 3). Mammalian cells involved in secretion discretely need to respond to the environment and move components within the cells and position them at appropriate locations for secretion (4, 5). This process involves force generation using Gibbs free energy of hydrolysis of adenosine triphosphate (ATP). The ATPase is most often myosin, a naturally occurring cellular ATPase known for its wide role in generation of cellular force (6). The nanomechanics of transport involve the necessary target cargoes, in association with myosin and track on actin filaments, which are ubiquitous cellular cytoskeletal scaffolds of metazoan cells (7). Cellular secretion encompasses multiple physiological systems operating on wide range of time scales including the processes of exocrine and endocrine glandular secretions and neuronal secretion in response to discrete electrical field stimulation, commonly referred to as neurotransmission (8, 9).

Identification of the PcrA DNA helicase reaction pathway by applying advanced targeted molecular dynamic simulations

PcrA DNA helicase uses the free energy of hydrolysis and binding of ATP to unwind double-stranded DNA (ds-DNA). There are two states of PcrA, termed the substrate and product complexes and, through the conformational changes between these two states, PcrA moves along ds-DNA and separates the two strands. In this study, two different methods, namely chain minimisation (CM, less reliable method) and auto targeted molecular dynamic (TMD) simulation (more reliable), were performed to generate two different initial reaction pathways between these two states, and then fixed root mean square distance (RMSD) TMD simulation was performed to optimise these two initial pathways. In general, the two optimised pathways share very similar major conformational changes, but are different in the minor motions. The potential energy profiles of the two improved pathways are generally similar, but the one generated by the improved TMD path is slightly lower. Considering the poor reliability of the initial path generated by CM and insignificant improvements of the auto-TMD path, our study suggests that fixed RMSD TMD simulation can generate reliable reaction pathways, but the different initial paths still have some influence on the detailed conformational analysis.

The Minimal Group Size for Globally Coordinated Stepping of Muscle Myosins Depends on ATP Hydrolysis Free Energy

In motility assays of muscle myosins, a distinct motile state emerges with increasing number of mechanically coupled myosin binding sites (N). This is explicable by coordinated myosin stepping (CMS): build-up of pre power stroke (PS) myosins is followed by a whole group PS and detachment cascade [1]. At low N, singular infrequent detachment cascades occur; at intermediate N, cascades group into bursts; for high N, interruptions between bursts disappear [1]. Here, we investigate how changing ATP hydrolysis free energy (ΔG) affects N-dependent emergence of CMS. We executed motility assays of muscle myosins and changed Pi concentration ([Pi]=0,1,2.5,5,15,30 mM, ionic strength adjusted by [KCl], [ATP]=2 mM, [ADP]=0.2 mM). Resolving actin sliding velocities by N [2] showed that increasing [Pi] increased the N at which bursts and continuous filament motion emerge. Lowering the rate of Pi release reproduced these observations in our detailed mechanochemical model of linearly elastic myosins mechanically coupled via an actin filament [1]. In this model, the rate of myosins' mechanical steps increases monotonically by ∼6 orders of magnitude dependent on the skew in myosin crossbridge strains (S). Plotting S and the fraction of myosin in the pre and the post PS state (n1,n2) reveals globally coordinated behavior that changes with N. N∼5: a quiescent state with high n1 dominates; N∼15: a quiescent state and cascading behavior with lowered n1 alternate; N∼30: cascading behavior dominates. An according continuous model shows two N-dependent stable steady states representing quiescence and cascading. Adding stochastic fluctuations in S lead to N-dependent cascade-like cycles. This suggests global coordination of myosins, which occurs above a minimal myosin group size that depends on ΔG.[1] Hilbert et al., Biophys J, 105(6):1466(2013) [2] Hilbert et al., PLoS Comp Biol, (2013).

Structure-based Molecular Simulations Reveal the Enhancement of Biased Brownian Motions in Single-headed Kinesin

Kinesin is a family of molecular motors that move unidirectionally along microtubules (MT) using ATP hydrolysis free energy. In the family, the conventional two-headed kinesin was experimentally characterized to move unidirectionally through “walking” in a hand-over-hand fashion by coordinated motions of the two heads. Interestingly a single-headed kinesin, a truncated KIF1A, still can generate a biased Brownian movement along MT, as observed by in vitro single molecule experiments. Thus, KIF1A must use a different mechanism from the conventional kinesin to achieve the unidirectional motions. Based on the energy landscape view of proteins, for the first time, we conducted a set of molecular simulations of the truncated KIF1A movements over an ATP hydrolysis cycle and found a mechanism exhibiting and enhancing stochastic forward-biased movements in a similar way to those in experiments. First, simulating stand-alone KIF1A, we did not find any biased movements, while we found that KIF1A with a large friction cargo-analog attached to the C-terminus can generate clearly biased Brownian movements upon an ATP hydrolysis cycle. The linked cargo-analog enhanced the detachment of the KIF1A from MT. Once detached, diffusion of the KIF1A head was restricted around the large cargo which was located in front of the head at the time of detachment, thus generating a forward bias of the diffusion. The cargo plays the role of a diffusional anchor, or cane, in KIF1A “walking.”

A Little Engine That Could: ATP-Powered Electrical Battery and Heater Inside Cells

A Little Engine That Could: ATP-Powered Electrical Battery and Heater Inside Cells

Elucidating the Molecular Origin of Hydrolysis Energy of Pyrophosphate in Water.

The molecular origin of the energy produced by the ATP hydrolysis has been one of the long-standing fundamental issues. A classical view is that the negative hydrolysis free energy of ATP originates from intramolecular effects connected with the backbone P-O bond, so called "high-energy bond". On the other hand, it has also been recognized that solvation effects are essential in determining the hydrolysis free energy. Here, using the 3D-RISM-SCF (three-dimensional reference interaction site model self-consistent field) theory that integrates the ab initio quantum chemistry method and the statistical mechanical theory of liquids, we investigate the molecular origin of hydrolysis free energy of pyrophosphate, an ATP analogue, in water. We demonstrate that our theory quantitatively reproduces the experimental results without the use of empirical parameters. We clarify the crucial role of water in converting the hydrolysis free energy in the gas phase determined solely by intramolecular effects, which ranges from endothermic, thermoneutral, to highly exothermic depending on the charged state of pyrophosphate, into moderately exothermic in the aqueous phase irrespective of the charged state as observed in experimental data. We elucidate that this is brought about by different natures of solute-water interactions depending on the charged state of solute species: the hydration free energy of low-charged state is mainly subjected to short-range hydrogen-bonds, while that of high-charged state is dominated by long-range electrostatic interactions. We thus provide unambiguous evidence on the critical role of water in determining the ATP hydrolysis free energy.

Molecular Simulation Study of Kinesin: Coupling between ATPase Domain Conformational Change and Mechanical Stepping

Conventional two-headed kinesin is a motor protein that moves unidirectionally by stepping in hand-over-hand manner (akin to human walking) along microtubule (MT) driven by ATP hydrolysis free energy. In the absence of MT, X-ray crystallography revealed primarily two conformations of the head, ATPase domain; “T” structure preferred with a bound ATP and “D” structure preferred with a bound ADP.However, relations among the ATPase conformations, stepping motion, and type of bound nucleotide are still rather unclear. Here, we investigated the coupling mechanism between the enzyme structure and mechanical stepping. For this purpose, we performed molecular dynamics simulations with coarse-grained structure-based models. In particular, to investigate structural preference between T and D in front and rear heads bound on MT, we applied multiple-basin energy landscape model (Okazaki et al., 2006).Through simulations, we found the followings. (1) Enzyme structure can regulate its affinity to MT by the difference in the contact surface area: “T” structure has higher affinity to MT than “D”, which is consistent with experiments. (2) The internal-strain between two heads can regulate the ATPase structural preference: The rear head with forward-directed neck-linker prefer T structure, while the front head with backward-directed neck-linker prefer D structure.

The significance of the free energy of hydrolysis of GTP for signal-transducing and regulatory GTPases

A large number of GTP/GDP binding proteins, which in general have intrinsic and/or stimulatable GTPase activity, have been identified in recent years and are involved in a wide range of cellular regulatory and signal transducing processes. A common property of these proteins is that they exist in what is generally described as an active form when GTP is bound and an inactive (resting) form when GDP is present. Thus, the intrinsic or stimulated GTPase activity of these ‘enzymes’ serves to turn off a signal or to terminate a regulated process. It has been suggested that these proteins, together with ATPases whose prime function is to convert the free energy of ATP hydrolysis into another form of energy or into energy-requiring chemical reactions should be grouped together under the heading of ‘energyases’. In this article, this suggestion is examined from the point of view of identifying the role of the free-energy of hydrolysis of GTP in the signal-transducing or regulatory process of the GTPases. It is concluded that there is a qualitative difference between ATPases and classical GTPases, in the sense that a quantitative relationship between the free-energy of GTP hydrolysis and the appearance of this energy in a different form cannot be directly defined. The significance of the high free energy of hydrolysis is that it allows efficient transition from the active to the inactive state of GTPases in spite of the tendency of the strong interaction of the GTP-bound form with a partner molecule (‘effector’), an essential feature of their mode of action, to stabilize the GTP-bound form.

Purine but Not Pyrimidine Nucleotides Support Rotation of F1-ATPase

The binding change model for the F(1)-ATPase predicts that its rotation is intimately correlated with the changes in the affinities of the three catalytic sites for nucleotides. If so, subtle differences in the nucleotide structure may have pronounced effects on rotation. Here we show by single-molecule imaging that purine nucleotides ATP, GTP, and ITP support rotation but pyrimidine nucleotides UTP and CTP do not, suggesting that the extra ring in purine is indispensable for proper operation of this molecular motor. Although the three purine nucleotides were bound to the enzyme at different rates, all showed similar rotational characteristics: counterclockwise rotation, 120 degrees steps each driven by hydrolysis of one nucleotide molecule, occasional back steps, rotary torque of approximately 40 piconewtons (pN).nm, and mechanical work done in a step of approximately 80 pN.nm. These latter characteristics are likely to be determined by the rotational mechanism built in the protein structure, which purine nucleotides can energize. With ATP and GTP, rotation was observed even when the free energy of hydrolysis was -80 pN.nm/molecule, indicating approximately 100% efficiency. Reconstituted F(o)F(1)-ATPase actively translocated protons by hydrolyzing ATP, GTP, and ITP, but CTP and UTP were not even hydrolyzed. Isolated F(1) very slowly hydrolyzed UTP (but not CTP), suggesting possible uncoupling from rotation.

Force Generation in RNA Polymerase

RNA polymerase (RNAP) is a processive molecular motor capable of generating forces of 25–30 pN, far in excess of any other known ATPase. This force derives from the hydrolysis free energy of nucleotides as they are incorporated into the growing RNA chain. The velocity of procession is limited by the rate of pyrophosphate release. Here we demonstrate how nucleotide triphosphate binding free energy can rectify the diffusion of RNAP, and show that this is sufficient to account for the quantitative features of the measured load-velocity curve. Predictions are made for the effect of changing pyrophosphate and nucleotide concentrations and for the statistical behavior of the system.

Myofibrils of skeletal muscle: the activity coefficient of orthophosphate.

In the myofibrils of skeletal muscle, at 22 degrees C, pH 7.1 and at the physiological protein osmotic pressure of 1.8 x 10(5) dynes/cm2, orthophosphate behaves quite ideally, the activity coefficient being 0.85. Under the same conditions and at saturation, 2.67 mumoles of orthophosphate are bound per gram of dry myofibrils, with a dissociation constant of 7 x 10(-5) molal. Work is in progress to determine the activity coefficients of adenine nucleotide analogues. This work is needed to assess the actual value of the free energy of hydrolysis of ATP in muscle.

Graded intracellular acidosis produces extensive and reversible reductions in the effective free energy change of ATP hydrolysis in a molluscan muscle.

Phosphorus nuclear magnetic resonance spectroscopy was used to evaluate the impact of experimental reductions of intracellular pH on in vitro preparations of the radula protractor muscle of the marine gastropod, Busycon canaliculatum. The intracellular pH of radula refractor muscle bundles superfused with buffered artificial sea water (pH = 7.8) was 7.29. It was possible to clamp muscle intracellular pH at various acidotic states by changing the superfusate to 5, 10, and 15 mmol.l-1 5,5-dimethyl-oxazolidine-2,4-dione in buffered artificial sea water (pH = 6.5). Consistent and temporally stable reductions of intracellular pH were achieved (intracellular pH = 6.98, 6.79, and 6.62, respectively). During the acidotic transitions, arginine phosphate concentrations decreased and inorganic phosphate concentrations increased in a reciprocal manner and remained essentially constant after the intracellular pH stabilized. The extent of changes in arginine phosphate and inorganic phosphate was directly proportional to the magnitude of the imposed acidosis. Total adenosine triphosphate concentrations remained unchanged in all treatments. However, the magnesium adenosine triphosphate to total adenosine triphosphate ratio declined in direct relation to the extent of the acidosis. Intracellular free Mg2+ fell incrementally with reduced intracellular pH. All of the above effects were rapidly reversed when the 5,5-dimethyl-oxazolidine-2,4-dione was washed out by changing the superfusate to buffered artificial sea water (pH = 7.8). Mg-adenosine diphosphate concentrations were calculated in all treatments using equilibrium constants for the arginine kinase reaction corrected for pH and intracellular free [Mg2+]. The metabolite, intracellular pH, and [Mg2+] data were used to estimate the effective free energy of hydrolysis of adenosine triphosphate (dG/d xi ATP) under most experimental conditions. Experimental acidosis resulted in dramatic reductions in dG/d xi ATP which were fully reversible upon wash-out of 5,5-dimethyl-dioxazolidine-2,4-dione and recovery to normal intracellular pH conditions. Acidosis resulted in net hydrolysis of arginine phosphate, likely via a complex mechanism involving enhancement of rate of adenosine triphosphate hydrolysis and/or inhibition of adenosine triphosphate synthesis.