PMB 103: METABOLISM: BASIC CONCEPTS AND DESIGN
Definition of terms; metabolism, bioenergetics and thermodynamics. * Laws of thermodynamics, free energy changes and standard free energy changes in biochemical reactions. * Phosphoryl group transfers and ATP; * Free-energy change for hydrolysis of ATP and other phosphorylated compounds and thioesters. * Role of ATP: phosphorylation, * pyrophosphorylation and adenylation, * assembly of informational macromolecules, * active transport and muscle contraction. * Biological oxidation-reduction reactions; * flow of electrons * dehydrogenations * redox potentials * electron carriers * dehydrogenases * Nature of metabolic reactions: anabolism, catabolism. * regulation of metabolism.

Scope of the course * (Review) the laws of thermodynamics and the quantitative relationships among free energy, enthalpy, and entropy. * describe the special role of ATP in biological energy exchanges. Consider the importance of oxidation-reduction reactions in living cells, the energetic of electron-transfer reactions, and the electron carriers commonly employed as cofactors of the enzymes that catalyze these reactions.

Reference Books 1. Lehninger, PPls of Biochemistry Fourth Edition David L Nelson and 2. Elementary Biophysics. An introduction. PK. Srivastave Alpha Science Oxford, UK 2005 3. Biophysics. V. Pattabhi and N. Gautham. Second Edition 2009 Alpha Science Oxford, UK

Introduction * Both plant and animals are made of cells. * The cells are highly complex and ordered structures but unstable and need constant use of energy to maintain their ordered structure and specific activity. * Both plant and animal cells require energy for growth and maintenance. * Lack of energy supply to the cell causes degrades into a random and disorganized state and consequently death. * Bioenergetics is the energy transformation process in living plants and animals. * It is the process through which they extract energy, how they use it to synthesize macromolecules and in performing other activities. * Transformations processes = the means by which energy from fuel metabolism or photosynthesis is coupled to a cell’s energy-requiring reactions. * The processes = carried out by highly co-ordinated net work of chemical reactions termed metabolism. * Solar = the primary source of energy for the living systems. * Green plants – use solar energy to synthesize the energy rich molecules that form the basic source of energy for all forms of life. * Living organisms = require energy to perform work, stay alive, grow and to reproduce. * Thus organisms carry out a variety of energy transductions, conversions of one form of energy to another. * They use the energy in fuels to synthesize the complex, highly ordered macromolecules from simple precursors. * They convert the chemical energy into concentration gradients and electrical gradients, into motion and heat, and into light * Biological energy transductions obey the same physical laws that govern all other natural processes.

Bioenergetics and Thermodynamics * Bioenergetics = quantitative study of the energy transductions in living cells and the nature and function of the chemical processes underlying the transductions.

Biological Energy Transformations Obey the Laws of Thermodynamics * Quantitative observations on the interconversion of different forms of energy led to the two fundamental laws of thermodynamics * The first law relates to conservation of energy and states that: for any physical or chemical change, the total amount of energy in the universe remains constant; energy may change form or it may be transported from one region to another, but it cannot be created or destroyed. * The second law of thermodynamics states that: the universe always tends toward increasing disorder: in all natural processes, the entropy of the universe increases. * Living organisms do not violate the second law; they operate strictly within it. * Reacting systems undergo a chemical or physical process mainly organisms, cells or two reacting compounds: reacting systems and its surroundings constitute the universe. * Living cells and organisms are open systems that exchange both material and energy with their surroundings and are never at equilibrium with their surroundings. * The constant transaction between the system and the surroundings shows how organisms create order within themselves while operating within the second law of thermodynamics. * Three thermodynamic quantities describe the energy changes in a chemical reaction:

1. Gibbs free energy * Gibbs free energy, G, expresses the amount of energy capable of doing work during a reaction at constant temperature and pressure. * When a reaction proceeds with the release of free energy the free-energy change, G, has a negative value and the reaction is said to be exergonic. * In endergonic reactions, the system gains free energy and G is positive.

Enthalpy * Enthalpy, H, is the heat content of the reacting system. * It reflects the number and kinds of chemical bonds in the reactants and products. * A chemical reaction that releases heat is termed exothermic the heat content of the products being less than that of the reactants and H has, by convention, a negative value. * Reacting systems that take up heat from their surroundings are endothermic and have positive values of H.

Entropy * Entropy, S, is a quantitative expression for the randomness or disorder in a system. * When the products of a reaction are less complex and more disordered than the reactants, the reaction is said to proceed with a gain in entropy. * The units of G and H are joules/mole or calories/mole. * Under biological systems, changes in free energy, enthalpy, and entropy are related to each other quantitatively by the equation in which G is the change in Gibbs free energy f the reacting system, H is the change in enthalpy of the system, T is the absolute temperature, and S is the change in entropy of the system. * By convention, S has a positive sign when entropy increases and H, as noted above, has a negative sign when heat is released by the system to its surroundings. * Either of these conditions, which are typical of favorable processes, tend to make G negative. In fact, * G of a spontaneously reacting system is always negative. * The second law of thermodynamics states that the entropy of the universe increases during all chemical and physical processes, but it does not require that the entropy increase take place in the reacting system itself. * The order produced within cells, as they grow and divide, is more than compensated for by the disorder they create in their surroundings in the course of growth and division. * Living organisms preserve their internal order by taking free energy from nutrients or solar, and return to their surroundings an equal amount of energy as heat and entropy.

Cells Require Sources of Free Energy. * Energy that cells use is free energy, which allows prediction of the direction of chemical reactions, exact equilibrium position, and the amount of work they can perform. * Heterotrophiles and autotrophs acquire free energy from nutrient molecules and absorption of the solar radiation. * The two kinds of cells transform free energy into ATP and other energy-rich compounds capable of providing energy for biological work. * Cell function depends largely on molecules, such as proteins and nucleic acids whose free energy of formation is positive * The molecules are less stable and more highly ordered than a mixture of their monomeric components. * To carry out thermodynamically unfavorable, energy-requiring (endergonic) reactions, cells couple these to other reactions that liberate free energy (exergonic reactions), so that the overall process is exergonic: the sum of the free energy changes is negative. * The usual source of free energy in coupled biological reactions is the energy released by hydrolysis of phosphoanhydride bonds such as those in ATP.

The Standard Free-Energy Change Is Directly Related to the Equilibrium Constant
The standard free-energy changes for some representative chemical reactions
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* Hydrolysis of esters, amides, peptides, and glycosides, rearrangements and eliminations, proceed with relatively small standard free-energy changes. In contrast, * Hydrolysis of acid anhydrides is accompanied by relatively large decreases in standard free energy. * Complete oxidation of organic compounds (glucose or palmitate to CO2 and H2O, results in very large decreases in standard free energy. * However, standard free-energy changes (as shown on Table above) indicate how much free energy is available from a reaction under standard conditions. * The positive value of G predicts that under standard conditions the reaction will tend not to proceed spontaneously (in the direction written). * The cellular reaction, the hydrolysis of ATP to ADP and Pi, is very exergonic:
ATP + H2O →ADP + Pi G -30.5 kJ/mol * In thermodynamic reactions, all that matters is the state of the system at the beginning of the process and at the end; the route between the initial and final states is immaterial. * Glycolysis, HMP and the TCA are important cellular pathways for producing ATP whose net or end products are additive and exergonic.

Phosphoryl Group Transfers and ATP
The ATP is the energy currency that links catabolism and anabolism as shown below

* Heterotrophic cells obtain free energy in a chemical form by the catabolism of nutrient molecules, and then use that energy to make ATP from ADP and Pi. * The ATP donates its chemical energy to endergonic processes e.g, * Synthesis of metabolic intermediates and macromolecules, transport of substances across membranes against concentration gradients, mechanical motion. * Energy donation from ATP involves covalent participation of ATP in the reactions resulting into conversion of ATP into ADP and Pi or and at times to AMP and 2 Pi * Large free-energy changes accompany hydrolysis of ATP and other high-energy phosphate compounds And in most cases * Energy donation by ATP involves group transfer, not simple hydrolysis of ATP. * There is a range of energy transductions in which ATP provides the energy mostly during biosynthesis of information-rich macromolecules, and transport of solutes across membranes, and motion produced by muscle contraction.
The Free-Energy Change for ATP Hydrolysis Is Large and Negative * Figure below summarizes the chemical basis for the relatively large, negative, standard free energy of hydrolysis of ATP. * .The hydrolytic cleavage of the terminal phosphoric acid anhydride (phosphoanhydride) bond in ATP separates one of the three negatively charged phosphates.

Figure (13-1). Chemical basis for the large free-energy change associated with ATP hydrolysis. 1) The charge separation that results from hydrolysis relieves electrostatic repulsion among the four negative charges on ATP. 2) The product inorganic phosphate (Pi) is stabilized by formation of a resonance hybrid, in which each of the four phosphorus–oxygen bonds has the same degree of double-bond character and the hydrogen ion is not permanently associated with any one of the oxygens. (Some degree of resonance stabilization also occurs in phosphates involved in ester or anhydride linkages, but fewer resonance forms are possible than for Pi.) 3) The product ADP2 immediately ionizes, releasing a proton into a medium of very low [H] (pH 7). A fourth factor (not shown) that favors ATP hydrolysis is the greater degree of solvation (hydration) of the products Pi and ADP relative to ATP, which further stabilizes the products relative to the reactants. * This relieves some of the electrostatic repulsion in ATP; the Pi (HPO42-) released is stabilized by the formation of several resonance forms not possible in ATP; and ADP2-, other direct product of hydrolysis ionizes, releasing H into a medium of very low [H+]. * Concentrations of the direct products of ATP hydrolysis are, in the cell, far below the concentrations at equilibrium (Table 13–5), thus mass action favors the hydrolysis reaction in the cell. * The hydrolysis of ATP is highly exergonic (G -30.5 kJ/mol), the molecule is kinetically stable at pH 7 because the activation energy for ATP hydrolysis is relatively high. * Rapid cleavage of the phosphoanhydride bonds occurs when catalyzed by an enzyme. * The free-energy change for ATP hydrolysis is -30.5 kJ/mol but the actual free energy of hydrolysis (G) of ATP in living cells is very different: the cellular concentrations of ATP, ADP, and Pi are not identical and are much lower than the 1.0 M of standard conditions. * Furthermore, Mg2 in the cytosol binds to ATP and ADP (Fig. 13–2), and for most enzymatic reactions that involve ATP as phosphoryl group donor, the true substrate is MgATP2- as shown below

* The relevant G is therefore that for MgATP2 hydrolysis. * G for ATP hydrolysis, in intact cells, usually Gp, is much more negative than G, ranging from 50 to 65 kJ/mol. * Gp is often called the phosphorylation potential.

Free Energies of Hydrolysis in Phosphorylated Compounds and Thioesters * The phosphate ester bond in `Phosphoenolpyruvate (PEP) undergoes hydrolysis to yield the enol form of pyruvate that tautomerize to keto form of pyruvate as shown below.

* Because the reactant (PEP) has only one form (enol) and the product (pyruvate) has two possible forms, the product is stabilized relative to the reactant. * This is the greatest contributing factor to the high standard free energy of hydrolysis of phosphoenolpyruvate: G 61.9 kJ/mol. * 1,3-bisphosphoglycerate (Fig. 13–4), contains an anhydride bond between the carboxyl group at C-1 and phosphoric acid. * Its hydrolysis is accompanied by a large, negative, standard free-energy change ( G =49.3 kJ/mol), which can, again, be explained in terms of the structure of reactant and products * When H2O is added across the anhydride bond of 1,3-bisphospho- glycerate, one of the direct products, 3-phosphoglyceric acid, can immediately lose a proton to give the carboxylate ion, 3-phosphoglycerate, which has two equally probable resonance forms as shown below * * Removal of the direct product (3-phosphoglyceric acid) and formation of the resonance-stabilized ion favor the forward reaction. * In phosphocreatine, the P-N bond can be hydrolyzed to generate free creatine and Pi as shown below

* The release of Pi and the resonance stabilization of creatine favor the forward reaction. * The standard free-energy change of phosphocreatine hydrolysis is again large, 43.0 kJ/mol. * In all phosphate-releasing reactions (as shown below), the several resonance forms available to Pi stabilize this product relative to the reactant, contributing to an already negative free-energy change. * Thioesters also have large, negative, standard free energies of hydrolysis and includes * Acetyl-coenzyme A, or acetyl-CoA (as shown below), is one of many thioesters important in metabolism. * * Their acyl group is activated for transacylation, condensation, or oxidation-reduction reactions. * Esters undergo less resonance stabilization than oxygen esters hence the difference in free energy between the reactant and its hydrolysis products, which are resonance-stabilized, is greater for thioesters than for comparable oxygen esters (as shown above).

* In both cases, hydrolysis of the ester generates a carboxylic acid, which can ionize and assume several resonance forms. * Thus acetyl-CoA hydrolysis has a large, negative G (31 kJ/mol).
ATP Provides Energy by Group Transfers (Not by Simple Hydrolysis) * A single reaction arrow such as ATP + H2O →ADP + Pi G30.5 kJ/mol represents a two-step process * Part of the ATP molecule, a phosphoryl or pyrophosphoryl group or the adenylate moiety (AMP) is transferred, to a substrate molecule or to an amino acid residue, into an enzyme, and covalently attached to the substrate or the enzyme raising its free-energy content. * Reactions of ATP appear as simple hydrolysis reactions in which water displaces Pi (or PPi), or that an ATP-dependent reaction is “driven by the hydrolysis of ATP.” * ATP hydrolysis only liberate heat, which cannot drive a chemical process.
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* The phosphate-containing moiety (transferred in the first step) is then displaced, generating Pi, PPi, or AMP. Thereafter, * ATP is able to participate covalently in enzyme-catalyzed reaction to which it contributes free energy. * Some processes involve hydrolysis of ATP/GTP, non-covalent binding of ATP/GTP then hydrolysis to ADP/GDP and Pi. * This provides energy that cycle proteins between two conformations, producing mechanical motion: e.g., muscle contraction, movement of enzymes along DNA and ribosomes along messenger RNA. * ATP -dependent reactions catalyzed by helicases, RecA protein, and topoisomerases involve hydrolysis of phosphoanhydrides. * Signalling pathways involve GTP-ases that hydrolyze GTP to drive conformational changes that terminate signals triggered by hormones or by other extracellular factors. * Phosphate compounds in cells consist of two groups, based on their standard free energies of hydrolysis as shown below

* “High-energy” compounds have a G of hydrolysis more negative than -25 kJ/mol; “low-energy” compounds have a less negative G. * Thus, ATP, with a G of hydrolysis of -30.5 kJ/mol, is a high-energy compound; glucose 6-phosphate, with a G of hydrolysis of 13.8 kJ/mol, is a low-energy compound. * Breaking of chemical bonds requires energy input. Besides, * Phosphorylated compounds are synthesized by coupling the synthesis to the breakdown of another phosphorylated compound with a more negative free energy of hydrolysis. E.g., * cleavage of Pi from PEP releases more energy than is needed to drive the condensation of Pi with ADP * Direct donation of a phosphoryl group from PEP to ADP is thermodynamically feasible:

* Notice that while the overall reaction above is represented as the algebraic sum of the first two reactions, the overall reaction is actually a third, distinct reaction that does not involve Pi; PEP donates a phosphoryl group directly to ADP. Thus, * Phosphorylated compounds are high or low phosphoryl group transfer potential based on the basis of their standard free energies of hydrolysis (as listed in Table above). * The phosphoryl group transfer potential of PEP is very high, that of ATP is high, and that of glucose 6-phosphate is low (Fig. 13–9). * Catabolism is directed toward the synthesis of high-energy phosphate compounds. * However, their formation is the means of activating a very wide variety of compounds for further chemical transformation. * Transfer of a phosphoryl group to a compound puts free energy into that compound, so that it has more free energy to give up during subsequent metabolic transformations: synthesis of G6P following phosphoryl group transfer from ATP. * Phosphorylation of glucose activates, or “primes,” it for catabolic reactions that occur in nearly every living cell. * ATP carry energy from high-energy phosphate compounds produced by catabolism to compounds such as glucose, converting them into more reactive species. * ATP thus serves as the universal energy currency in all living cells. * In aqueous solution ATP is thermodynamically unstable and is therefore a good phosphoryl group donor, it is kinetically stable. * ATP requires huge activation energies (200 to 400 kJ/mol) for uncatalyzed cleavage of its phosphoanhydride bonds. Hence * This limits of ATP to spontaneously donate phosphoryl groups to water and hundreds of other potential acceptors in the cell. * Presence of specific enzymes lowers the energy of activation thereby enabling phosphoryl group transfer from ATP. Hence, * Cells regulate the disposition of the energy carried by ATP by regulating the various enzymes that act on it.

ATP Donates Phosphoryl, Pyrophosphoryl, and Adenylyl Groups * ATP reactions - generally SN2 nucleophilic displacements (p. II.8) where the nucleophile, e.g., O2 of an alcohol (ROH), carboxylate (RCOO-), N of creatine or of the side chain of arginine or histidine. * Each of the three phosphates of ATP is susceptible to nucleophilic attack (Fig. 13–10): each position of attack yields a different type of product as shown below.

* Nucleophilic attack by an alcohol (ROH) on the phosphate (Fig. 13–10a) displaces ADP and produces a new phosphate ester. * Radiolabelled reactants showed the bridge O2 in the new compound is derived from the alcohol, not from ATP; the group transferred from ATP is a phosphoryl , not a phosphate (see above Figure) * Phosphoryl group transfer from ATP to glutamate (Fig. 13–8) or to glucose involves attack at the  position of the ATP molecule. * Attack at the  phosphate of ATP displaces AMP and transfers a pyrophosphoryl (not pyrophosphate) group to the attacking nucleophile (Fig. 13–10b). * For example, the formation of 5′-phosphoribosyl-1-pyrophosphate, an intermediate in nucleotide synthesis, results from attack of an OOH of the ribose on the phosphate. * Nucleophilic attack at the  position of ATP displaces PPi and transfers adenylate (5 -AMP) as an adenylyl group (Fig. 13–10c); the reaction is an adenylylation. * Hydrolysis of phosphoanhydride bond releases considerably more energy (~46 kJ/mol) than hydrolysis of the -bond (~31 kJ/mol) (Table 13–6). * The PPi formed as a byproduct of the adenylylation is hydrolyzed to two Pi by inorganic pyrophosphatase, releasing 19 kJ/mol. * Both phosphoanhydride bonds of ATP are split in the overall reaction. * Adenylylation reactions = thermodynamically very favorable. * ATP = drive unfavorable metabolic reaction with adenylylation being the mechanism of energy coupling, e,g, in fatty acid activation being good example of energy-coupling strategy. * Activation of FAs= for energy-yielding oxidation or for synthesis of more complex lipids—is the formation of its thiol ester (see Fig. 17–5). * Condensation of a FA with coenzyme A is endergonic whereas formation of fatty acyl–CoA is exergonic due to stepwise removal of two phosphoryl groups from ATP.

Assembly of Informational Macromolecules Requires Energy * Energy is required both for the condensation of monomeric units and for the creation of ordered sequences. * This occurs when simple precursors are assembled into high molecular weight polymers such a DNA, RNA or proteins. * The precursors for DNA and RNA synthesis are nucleoside triphosphates, and polymerization is accompanied by cleavage of the phosphoanhydride linkage between the  and  phosphates, with the release of PPi as shown below (Fig. 13–11).

* * The moieties transferred to the growing polymer in these reactions are adenylate (AMP), guanylate (GMP), cytidylate (CMP), or uridylate (UMP) for RNA synthesis, and their deoxy analogs (with TMP in place of UMP) for DNA synthesis. * The activation of amino acids for protein synthesis involves the donation of adenylate groups from ATP, * Several steps of protein synthesis on the ribosome are also accompanied by GTP hydrolysis. * In all these cases, the exergonic breakdown of a nucleoside triphosphate is coupled to the endergonic process of synthesizing a polymer of a specific sequence. * Energy is required both for the condensation of monomeric units and for the creation of ordered sequences. ATP Energizes Active Transport and Muscle Contraction * ATP can supply the energy for transporting an ion or a molecule across a membrane into another aqueous compartment where its concentration is higher as shown below (see Fig. 11–36). *

* Transport processes are major consumers of energy; in human kidney and brain, for example, as much as two-thirds of the energy consumed at rest is used to pump Na and K across plasma membranes via the Na+ K+ ATPase. * The transport of Na+ and K+ is driven by cyclic phosphorylation and dephosphorylation of the transporter protein, with ATP as the phosphoryl group donor as shown below (see Fig. 11–37).

* Na+-dependent phosphorylation of the Na+ K+ ATPase forces a change in the protein’s conformation, and K+-dependent dephosphorylation favors return to the original conformation. * Each cycle in the transport process results in the conversion of ATP to ADP and Pi , and it is the free-energy change of ATP hydrolysis that drives the cyclic changes in protein conformation that result in the electrogenic pumping of Na and K. * Note that in this case ATP interacts covalently by phosphoryl group transfer to the enzyme, not the substrate. * In the contractile system of skeletal muscle cells, myosin and actin are specialized to transduce the chemical energy of ATP into motion (see Fig. 5–33). * ATP binds tightly but noncovalently to one conformation of myosin, holding the protein in that conformation. * When myosin catalyzes the hydrolysis of its bound ATP, the ADP and Pi dissociate from the protein, allowing it to relax into a second conformation until another molecule of ATP binds. * The binding and subsequent hydrolysis of ATP (by myosin ATPase) provide the energy that forces cyclic changes in the conformation of the myosin head. * The change in conformation of many individual myosin molecules results in the sliding of myosin fibrils along actin filaments (see Fig. 5–32), which translates into macroscopic contraction of the muscle fiber. * As we noted earlier, this production of mechanical motion at the expense of ATP is one of the few cases in which ATP hydrolysis per se, rather than group transfer from ATP, is the source of the chemical energy in a coupled process.