Free Energy Of Hydrolysis Research Articles

Control involving metabolism and gene expression

Control of DNA supercoiling by the free-energy of hydrolysis of ATP that involves gene expression is analyzed in terms of three levels of unconnected metabolic pathways. These are synthesis and breakdown of topoisomerase mRNAs, synthesis and breakdown of topoisomerase proteins and supercoiling and relaxation of DNA. The so-called square-matrix method previously developed for the control of metabolic pathways, is extended to deal with this hierarchical control system. It turns out that also in this case, the matrix of control coefficients is equal to the inverse of the so-called elasticity matrix, which contains all relevant elasticity coefficients as well as information about the structure and connectedness of the pathways involved. For a simpler case of a hierarchy of two systems, we demonstrate that the explicit matrix inversion method may be replaced by an implicit in which the regulatory effects that run through the other level are described by an additional elasticity coefficient which may then be treated as if local.

Meaning of energetic parameters.

High energy phosphates (HEP: ATP & phosphocreatine (PCr)) are to be considered under qualitative (free energy of hydrolysis) and quantitative (concentration) aspects. Energy charge is also a (normalized) concentration related parameter. Owing to high turnover rates concentration (HEP stores) provides sufficient energy only for 10 to 20 s in the case of interruption of supply. Utilization (without immediate supply) leads to rapid drop of free energy which--owing to the CK reaction--is indicated by rapid drop of PCr and the respective ratios. Critical reduction of free energy appears to be prevented by various protective mechanisms reflected in phenomena of early hypoxic failure, hibernating and stunning.

Energetics and the design principles of the Na/K-ATPase

Following on the pioneering analysis by Pickart and Jencks of the energetics of the calcium pump, the present mini review attempts a similar analysis of the somewhat more complicated sodium--and potassium--activated ATPase pump-enzyme. The analysis is based on the measurements of the rate-constants of the individual steps in the enzymic reaction which brings about pumping and uses data assembled by Stürmer et al., in the laboratory of Peter Läuger in Konstanz. The aim of such an analysis is to calculate the overall free energy released on ATP hydrolysis and to apportion this energy among the successive steps of the pump-enzyme reaction. We calculate the so-called basic free energy changes that take into account the prevailing ligand and ion concentrations, rather than the standard-state free energies that refer to 1 M concentrations of these ions and ligands. Using appropriate values of the ion and ligand concentrations in cardiac muscle, the available free energy which can be released from the hydrolysis of ATP (at 20 degrees C) comes out at 575 mV. Following a complete cycle of pumping, 371 mV of this free energy are found stored in the sodium and potassium ion gradients. The remaining 204 mV from the free energy of hydrolysis of ATP are lost to the ATP system. This part of the energy, that had been transduced into the concentration gradient of sodium, has presumably been used in the living cell to drive the co- and counter-transport (symport and antiport) of ions and metabolites in secondary transports. The free energy changes are pretty evenly apportioned along the various steps in the pumping cycle. The steps that one might naively have thought to be "powered", such as the step in which covalently bound phosphate is transformed from a high-energy to a low-energy state, or the step in which sodium is released into the phase containing a high concentration of sodium, show some of the lowest drops of free energy, 61 mV and 27 mV, respectively. The most surprising step in the overall reaction of ATP hydrolysis and synthesis is the phosphorylation of the protein from inorganic phosphate with formation of the acylphosphate bond. The stabilization of the acylphosphate bond presumably arises from ionic interactions between the covalently bound phosphate itself and appropriate groupings on the enzyme. ATP formation on the F0F1 ATPase (the F-type ATPase) is in an analogous way stabilized in the first place by phospho-ligand/enzyme interactions.

Crystal and Molecular Structure of Phosphoenolpyruvate Salts

Abstract Phosphoenolpyruvate (PEP) plays central role in the storage and transfer of energy in biological systems. Its free energy of hydrolysis (-58.5 kJ mol−1) is the highest known for monoesterified phosphate esters. Depending on pH PEP can occur as monoanion, dianion and trianion or as tri-basic acid. Studying PEP salts in different crystal forms provides additional information on its molecular flexibility, phosphate group conjugation with the enolpyruvate residue and kind of binding to different cations.

Changes in Cellular Bioenergetic State Following Graded Traumatic Brain Injury in Rats: Determination by Phosphorus 31 Magnetic Resonance Spectroscopy

Phosphorus 31 magnetic resonance spectroscopy (31P MRS) was used to study noninvasively the intracellular free Mg2+ concentration and cellular bioenergetic state of rat brain in vivo before and after fluid percussion-induced traumatic brain injury of graded severity. Brain injury was induced at four levels: low (1.0 +/- 0.5 atm); moderate (2.1 +/- 0.4 atm); high (3.9 +/- 0.9 atm); and severe (5.9 +/- 0.7 atm). Prior to injury, mean intracellular values for all groups (n = 24; mean +/- SE) were as follows: pH = 7.11 +/- 0.03; free [Mg2+] = 0.99 +/- 0.07 mM; cytosolic [ADP] = 25.2 +/- 0.8 nmol/g wet weight; cytosolic [AMP] = 0.29 +/- 0.02 nmol/g wet weight; cytosolic phosphorylation potential = 118.5 +/- 3.1 X 10(3) M-1; free energy of ATP hydrolysis = 62.11 +/- 0.04 kJ/mole; and energy charge = 0.99 +/- 0.01. Following every level of injury, there were decreases in intracellular free Mg2+ concentration, and alterations in the intracellular pH. These posttraumatic changes in Mg2+ and pH induced shifts in the equilibrium constants of the creatine kinase, adenylate kinase, and ATPase reactions, resulting in alterations in [ADP], [AMP], cytosolic phosphorylation potential, and free energy of hydrolysis, but not in the energy charge. The alterations in cytosolic phosphorylation potential following trauma were linearly correlated with the changes in intracellular free Mg2+ concentration. None of the individual bioenergetic parameters could be correlated with the severity of injury over the entire injury range; however, an association between cytosolic phosphorylation potential and reversibility of injury was apparent. These results suggest that reductions in cellular bioenergetic state following traumatic brain injury occur through a posttraumatic decrease in the cells' capacity for oxidative phosphorylation, which itself may be directly related to the intracellular free Mg2+ concentration.

Role of Water on the Free Energy of Hydrolysis of Pyrophosphate

This chapter discusses the role of water on the free energy of hydrolysis of pyrophosphate. The energy of hydrolysis of pyrophosphate and other compounds is determined by the difference in solvation energies of reactant and products. The chapter discusses the experiment where 32Piwas obtained from the Brazilian Institute of Atomic Energy and purified and the synthesis of pyrophosphate was assayed by measuring the formation of radioactive pyrophosphate from 32Pi. The specific activity was 500 units/mg of enzyme. The data presented in the chapter can be interpreted according to the concept of solvation energy. The finding that in mixed solvents there is a significant decrease in the Kobsfor pyrophosphate hydrolysis strongly suggests that organization of water around reactants and products plays a major role in determining the thermodynamic parameters of pyrophosphate hydrolysis. Water possesses the most cooperative structure of all common solvents, but it is destroyed by organic cosolvents. The chemical reactivity of water molecules is scarcely affected by cosolvents. However, the reactions that depend on solvent structure change markedly with cosolvent addition.

Effect of ATP/ADP/phosphate potential on the maximal steady-state uptake of Ca2+ by skeletal sarcoplasmic reticulum.

The ability of the Ca2+-Mg2+ ATPase pump of skeletal SR to produce and maintain a Ca2+ gradient was studied as a function of the ATP/ADP/Pi ratio. The internal free Ca2+ concentration [Ca2+]i was monitored by changes in fluorescence of CTC. Increasing ADP concentrations in the medium reduce the maximal [Ca2+]i concentration achieved. The inclusion or the omission of 4×10−4 M Pi or doubling the absolute ATP and ADP concentrations at a constant ATP/ADP ratio does not affect the level obtained. The level depends primarily on the ATP/ADP ratio. The [Ca2+] concentration shows a 1.5 power dependence on the ATP/ADP ratio. Further, [Ca2+]i achieved at steady state does not depend on whether the pump had been working in the forward or the reverse direction prior to testing. Analysis shows that the levels of Ca2+ achieved are much lower than the levels predicted thermodynamically under the assumption of ideal coupling between Ca2+ transport and ATP hydrolysis with a stoichiometry of 2:1. Under this condition the “osmotic” energy of the [Ca2+]i/[Ca2+]o ratio was shown to be 48% as large as the free energy of hydrolysis of ATP, giving an overall thermodynamic efficiency of 48%. Analysis shows that maximal steady-state uptake is determined by the balance between the rates of uptake by the pump and rates of leak processes (intrinsic or extrinsic to the pump). Comparison with other studies shows that the [Ca2+]i achieved results in trans-inhibition of the pump by tying up the Ca2+ translocator in the inwardly oriented phosphorylated form. The absence of an effect of Pi can be taken as evidence that the dissociation of Ca2+ from the inwardly oriented translocator on the phosphoylated enzyme must precede the dephosphorylation of the enzyme.

Bioenergetic aspects and polymer length distribution in steady-state head-to-tail polymerization of actin or microtubules.

Wegner's theory of steady-state head-to-tail polymerization of actin (or microtubules) is extended somewhat in order to show the explicit role of the ATP (or GTP) free energy of hydrolysis (X) in the steady-state kinetics. The monomer flux and the ATP flux can both be expressed in terms of X and rate constants of the model. Both fluxes approach zero as X leads to 0 (by variation of the concentrations of ATP, ADP, and Pi); this limit corresponds to ATP equilibrium. The dependence of rate constants on these concentrations is examined. Free energy levels of the monomer kinetic cycle and the rate of free energy dissipation are discussed. The steady-state polymer length distribution is derived for a special case.

Design principles for active transport systems

We have previously described simple models for active transport and have derived steady state equations for the unidirectional flux of substrate in terms of a minimal set of kinetic parameters. Here we consider how to maximize the pumping rate of a carrier-enzyme through the optimal utilization of the ATP hydrolysis reaction. The equations for net flux contain rate constants and dissociation constants and these determine the maximum velocities and affinities measured in transport kinetic analysis. It is assumed that the rate constants can evolve to the diffusion limited rate of substrate binding as has apparently occurred in the enzyme triosephosphate isomerase (Knowles & Albery, 1977). The dissociation constants of the rate limiting intermediates fit the affinities for substrates on different sides of the membrane and are dependent on the basic free energy levels (Hill, 1976) of the carrier substrate system. From our analysis it is clear that there are three ways to design a system with optimal affinities and that the choice is linked to the sequence of substrate binding. It is possible to use free energy differences of isomerization (Boyer, 1975) or ligand-ligand interactions (Weber, 1975) both of which have been described previously, but which are incorporated here into a unified treatment. A third possibility is to couple the binding step of a transported ligand to the progress of a chemical reaction as might occur, for example, if Na + must be bound before the carrier can be phosphorylated. In this way the free energy of hydrolysis can be used not only to drive the overall pumping reaction, but also to fix differentially the affinity for substrate on either side of the membrane, as required for rapid pumping.

Reversibility of adenosine 3':5'-monophosphate-dependent protein kinase reactions.

Using a homogeneous enzyme from rabbit skeletal muscle, it has been demonstrated that the cyclic adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase reaction is reversible. In addition to the phosphorylated protein substrate, the reverse reaction requires Mg2+, ADP, and cyclic AMP when the holoenzyme is used as the source of enzyme. It is independent of cyclic AMP when the catalytic subunit of the protein kinase is used. The optimum pH for the reverse reaction with 32P-labeled casein as the substrate is 5.7, essentially the same as that for the forward reaction. Among the nucleotide subtrates tested, ADP serves as the best phosphoryl group acceptor. The Km of the enzyme for ADP is 3.3 mM and that for 32P-casein is 1.7 mg/ml. The equilibrium constant at 30 degrees is approximately 0.042 at a magnesium concentration of 10 mM and a pH of 6.9. This result indicates that the free energy of hydrolysis (deltaG0obs) of the phosphorylated protein substrate is relatively high, i.e. approximately -6.5 kcal/mol under these conditions.

Effect of ADH, aldosterone, ouabain, and amiloride on toad bladder epithelial cells.

Effect of ADH, aldosterone, ouabain, and amiloride on toad bladder epithelial cells.

Purification and Properties of Bacillus subtilis Nucleoside Diphosphokinase

Abstract Bacillus subtilis nucleoside diphosphokinase was purified 1,100-fold to apparent homogeneity (a single band upon disc gel electrophoresis). Gel filtration through Sephadex G-200 indicated an approximate molecular weight of 100,000. The corrected Michaelis constants for the various nucleoside triphosphate substrates ranged from 0.10 mm for GTP to 0.42 mm for CTP. Deoxyribonucleoside triphosphates as well as ribonucleoside triphosphates served as substrates for the enzyme. Mn2+ and Mg2+ both were able to fulfill the divalent cation requirements for enzyme activity. Mn2+ was maximally activating at 1 mm while Mg2+ was maximally activating at 10 mm. The purified enzyme reacts with [γ-32P]ATP yielding a phosphorylated enzyme in which the phosphate group is bound via a 1-phosphohistidine bond. The equilibrium between the free and phosphorylated enzyme form favors the free form, indicating that the 1-phosphohistidine bond has a free energy of hydrolysis more negative than the terminal ATP bond. Initial velocity and product inhibition kinetic studies suggest a ping pong reaction mechanism (the first product dissociates before the second substrate combines with the enzyme). This is consistent with the postulation that the phosphorylated protein associated with enzyme activity is an intermediate in the enzymatic reaction.

The Free Energy of Hydrolysis of Adenosine to Inosine and Ammonia

Abstract The equilibrium constant for formation of adenosine from inosine and ammonia has been measured directly in the presence of adenosine deaminase at several pH values. The apparent free energy of hydrolysis of adenosine at pH 7.0 is -5.4 kcal per mole, and the free energy of hydrolysis to free ammonia and inosine is -2.2 kcal per mole, at ionic strength 1.0, 25°, with water concentration taken as 1. These values, which are comparable with indirect summations of thermochemical and equilibrium data for adenine and adenylic acid, are discussed in relation to earlier values for the hydrolysis of arginine and of amino acid amides.

A Determination of the Standard Free Energy of Hydrolysis of Pyrophosphate by Thermal Measurements. I. The Heat of Hydrolysis

A Determination of the Standard Free Energy of Hydrolysis of Pyrophosphate by Thermal Measurements. I. The Heat of Hydrolysis

THE FREE ENERGY OF HYDROLYSIS OF PHOSPHORYL-PHOSPHATASE.

THE FREE ENERGY OF HYDROLYSIS OF PHOSPHORYL-PHOSPHATASE.

The Free Energy of Thiol Ester Hydrolysis

The Free Energy of Thiol Ester Hydrolysis

The free energy of hydrolysis of adenosine triphosphoric acid.

DURING the past decade, the value of the energy of adenosine triphosphate breakdown has several times been subject to revision. The more precise, though indirect, calculations1 resulted in lower values (7–9 kcal./mol.) than the earlier results (11–13 kcal./mol.).

DETERMINATION OF THE EQUILIBRIUM OF THE HEXOKINASE REACTION AND THE FREE ENERGY OF HYDROLYSIS OF ADENOSINE TRIPHOSPHATE

DETERMINATION OF THE EQUILIBRIUM OF THE HEXOKINASE REACTION AND THE FREE ENERGY OF HYDROLYSIS OF ADENOSINE TRIPHOSPHATE

The free energy of hydrolysis of p-nitrophenyl phosphate.

The free energy of hydrolysis of p-nitrophenyl phosphate.