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Bioenergetics and High Energy Compounds Dr. Sugandhika Suresh Department of Biochemistry • Bioenergetics is the study of how organisms manage their energy resources • Energy is the capacity to cause change • It exists in various forms, some of which can perform work – Chemical energy is potential energy available for release in a chemical reaction • Energy can be converted from one form to another Summary of Animal Metabolism Hydrolyse Monosaccharides, Amino acids, Fatty acids Food CHO, Protein, Fat Anabolism Biosynthesis Growth, Substances Cell ATP, NADH Energy ATP CO2 Movement (mechanical) Muscle Active Transport (Osmotic) O XI DI S E Bioluminescence (Light Emission) Electrical energy Nerve Transmission • The free-energy change of a reaction tells us whether the reaction occurs spontaneously • A living system‟s free energy is energy that can do work when temperature and pressure are uniform, as in a living cell Free-Energy Change, G • The change in free energy (∆G) during a process is related to the change in enthalpy, or change in total energy (∆H), and change in entropy (T∆S): • Enthalpy will tell us the heat content (energy) of a system • Entropy will tell us whether a process is favourable (spontaneous) For any process (A B) at constant pressure and temperature The free energy change is defined as: ∆G = ∆H – T∆S (T = absolute temperature) Free Energy and Metabolism • The concept of free energy can be applied to the chemistry of life‟s processes • Reactions in a closed system eventually reach equilibrium and then do no work • Cells are not in equilibrium; they are open systems experiencing a constant flow of materials • A catabolic pathway in a cell releases free energy in a series of reactions A reaction can occur spontaneously (is favoured) only if ∆G is negative If: ∆G is negative (-) • the process is exergonic • the reaction proceeds with the release of free energy • the reaction will be thermodynamically favourable in the direction written LE 8-6a Free energy Reactants Amount of energy released (G < 0) Energy Products Progress of the reaction Exergonic reaction: energy released A reaction cannot occur spontaneously (is not favoured) if ∆G is positive If: ∆G is positive (+) • the process is endergonic • an input of free energy is required to drive the reaction • the reaction will be thermodynamically unfavoured (reverse process is favoured) LE 8-6b Free energy Products Energy Reactants Progress of the reaction Endergonic reaction: energy required Amount of energy required (G > 0) A system is at equilibrium and no net change can take place if ∆G is zero If: ∆G is zero (0) •The process is at equilibrium •No net flow in either the forward or the reverse direction •Neither process is favoured Most of these metabolic reactions are not spontaneous • •(i.e., they are accompanied by a positive change in free energy, ΔG>0) and do not occur without some other source of free energy. • Hence, the body needs some sort of "free-energy currency," a molecule that can store and release free energy when it is needed to power a given biochemical reaction. Just as purchasing transactions do not occur without monetary currency, reactions in the body do not occur without energy currency High Energy Compounds • Compounds with a high energy bond • If the bond is hydrolysed – chemical energy is made available • Less favourable reactions can be driven forward • ATP – most commonly encountered The free energy change for the hydrolysis of ATP is large and negative •This equilibrium lies so far to the right that ATP hydrolysis can be considered essentially irreversible •compounds that can undergo reactions with a resulting large negative free energy change (like ATP) are used as shuttles of free energy in the cell •(the bonds are said to contain potential transfer energy) • Because the free-energy changes of sequential reactions are additive; • Any phosphorylated compound can be synthesized by coupling the synthesis to the breakdown of another phosphorylated compound with a more negative free energy of hydrolysis (1) (2) Hydrolysis of PEP: Phosphorylation of ADP: PEP ADP + Pi Coupled phosphorylation of ADP by PEP: PEP + ADP pyruvate + Pi ATP ∆G o’ = - 62 kJ/mol ∆G o’ = + 31 kJ/mol pyruvate + ATP ∆G o’ = -31 kJ/mol cleavage of Pi from phosphoenolpyruvate (PEP) releases more free energy Coupling of biochemical reactions These two reactions share an intermediate (Pi) and may be expressed as sequential (coupled) reactions Glucose + Pi ATP + H20 ATP + glucose glucose 6- phosphate ADP + Pi ∆Go’ = + 13.8 kJ/mol ∆Go’ = - 30.5 kJ/mol ADP + glucose 6-phosphate ∆Go’ = -16.7 kJ/mol The energy stored in the bonds of ATP is used to drive the synthesis of glucose 6phosphate even though its formation from glucose and phosphate is endergonic In thermodynamic calculations, all that matters is the state of the system at the beginning of the process and its state at the end Note the range of potential transfer energy of these compounds (-60 to –10 ∆Go’ kJ/mol ) This indicates that some of these phosphate hydrolysis reactions are very high energy processes while others are not. Q: WHY do some compounds have a higher free energy of hydrolysis? It’s not the phosphate bonds themselves, but rather some property of both the reactants and products in the reactions that contribute to the differences in free energy The free energy of phosphate hydrolysis of ATP when compared to other molecules is at the middle. This allows ATP to accept energy from high energy donor compounds & to act as a donor for low energy phosphate acceptor. Functions carried out by ATP • Active transport - molecules & ions • Mechanical work – muscle contraction & other cellular movement • Biosynthesis of macromolecules (ATP serve as the immediate donor of free energy in biological systems) Compounds with equal free energy of hydrolysis 1. Nucleoside triphosphates/ Deoxy nucleoside triphosphates: GTP ATP CTP UTP (PPi bonds present in all compounds) Nucleoside diphosphate forms: ADP GDP CDP UDP (ATP + GDP GTP + ADP) Compounds with more Energy than ATP SUPER HIGH ENERGY COMPOUNDS 2. Creatine phosphate (CP) store of free energy in muscle CP + ADP ATP + creatine creatine kinase 3. PEP (glycolysis) PEP + ADP ATP + pyruvate ATP – Uses in Medical Context Active Transport •Absorption •HCl •I- of glucose in intestine secretion in stomach Pump in thyroid Muscle Contraction Energy in ATP Helix Random coil of Myosin Head In all cases ATP Energy = 7 -14 kcal.mole-1 ATPase ADP + Pi 27 4. NuDP Sugars (activated molecules) • UDP glucose • CDP glucose • ADP glucose • UDP galactose 5. Glucose derivative polymers Biosynthesis NuDP Bases • CDP choline Biosynthesis of phospholipids All involved in Biosynthesis = Anabolism 6. Other Coenzyme A Esters •Succinyl Co A •Malonyl CoA Cofactors Dr. Sugandhika Suresh Dept. of Biochemistry 30 • Small non-protein helpers of enzymes • Enzyme is not biologically active without the cofactor • Without the cofactor, the enzyme is known as the apo enzyme 31 • Apo enzyme = enzyme – (minus) prosthetic group Holo enzyme = enzyme + all cofactors = core enzyme + coenzyme eg: RNA polymerase + σ factor cofactor 32 Eg. carboxypeptidase 33 Cofactors (small non – protein units) Loosely bound Inorganic Mn2+ Mg2+ Organic Cosubstrate Coenzyme Tightly bound Prosthetic groups Organic Inorganic 34 Coenzymes • Heat stable, low molecular weight organic compounds • Gets transiently associated with the enzyme during enzyme activity • Mostly linked to the enzyme by non-covalent forces. Prosthetic group • Form tight covalent bonds, with the enzymes –. 35 Coenzymes • In oxidation-reduction reactions, coenzymes often remove electrons from the substrate and pass them to different enzymes. • In this way, coenzymes serve to carry energy in the form of electrons (or hydrogen atoms) from one compound to another. • Many coenzymes are derived from the vitamin B complex. 36 37 • Co-substrates: – ATP – NAD+ – NADP+ – FMN – FAD Changes at the end of a reaction 38 Co-substrates An example of a Co-substrate reaction (i) Glucose + ATP G6P + ADP gives energy (ii) Lactate + NAD+ NADH + H+ + PYRUVATE gives reducing power In both cases, co-substrate is not the same, at the end of the reaction. 39 Prosthetic groups Inorganic prosthetic groups Organic prosthetic groups Ion Enzyme Co2+ Carboxypeptidase Zn2+ Many enzymes Se Glutathione reductase Cu2+ Caeruloplasmin Haem Hb, Mb, Cytochromes (Porphyrin) CHO Glycoproteins Lipids Lipoproteins 40 Metabolism – All the Biochemical Pathways in Cells Metabolism Anabolism Catabolism Anabolism Catabolism 1. Energy using 1. Energy yielding 2. Reductive 2. Oxidative 3. Biosynthetic 3. Degradative 41 Metabolism: the highly integrated network of chemical reactions by which living cells grow & sustain themselves. The network is composed of two major types of pathways: 1) anabolic p.w (anabolism) 2) catabolic p.w (catabolism) 42 Anabolism: Process by which large molecules are synthesized using smaller molecules and energy stored in adenosine triphosphate (ATP) Catabolism: Process by which the nutrients & cell constituents are degraded to produce energy (as ATP) & also raw materials for anabolic reactions 43 Catabolism and anabolism 44 2 metabolic networks have 3 major functions: (1) extract energy from nutrients (2) synthesize building blocks that make up large molecules of life: proteins, fats, carbohydrates, nucleic acids & combinations of these substances (3) synthesize & degrade molecules required for special functions in the cell 45 Anabolic & catabolic reactions are enzymatic reactions organized into multi-step pathways (metabolic pathways). • Reaction sequences are composed of many enzymatic reactions, each one creating a product that becomes the substrate for the subsequent enzyme. (Enz – lower activation energy) 46 Metabolic pathway E - enzyme E1 E2 A B C E3 E4 D E5 E F metabolic intermediates or metabolites • Most reactions of anabolic or catabolic pathways are reversible with few unidirectional or irreversible reactions which control the pathway. 47 1. Metabolic pathways are irreversible. irreversible reaction (s) of a pathway makes the path way irreversible 2. Catabolic & anabolic pathways differ Catabolic p.w C A B Anabolic p.w X Y Why ? 48 A B exergonic (free energy released) - ΔG B A endergonic (free energy required) + ΔG The two pathways differ at least in one reaction Independent control (regulation ) of the two pathways 49 3. Every metabolic pathway has a committed step • an irreversible reaction early in the pathway (exergonic) (ΔG – ve) commits the intermediate to continue down the pathway • Most reactions of a pathway function close to equilibrium 50 4. All metabolic pathways are regulated by laws of supply & demand Most are controlled by regulating the enzymes that catalyze rate limiting steps “POINTS OF CONTROL” First committed step often is one of the rate limiting steps. Prevent unnecessary synthesis of metabolites further down the pathway 51 Eg: a) allosteric control feed back inhibition E1 E2 E3 A B C D E4 E E5 F b) covalent modification glycogen phosphorylase 5. Metabolic pathways occur in specific cellular locations (compartmentation) 52 Catabolism: different proteins, polysaccharides & lipids are broken down into relatively few catabolic end products. Convergent 53 Stages in the extraction of energy from foodstuff i) Large molecules smaller units. ii) small molecules into a few simple units that play a central role in metabolism. Some ATP generated. iii) TCA cycle & oxidative phosphorylation – FINAL COMMON p.w in oxidation of fuel molecules. > 90% of ATP from 54 food released int Anabolism: relatively few biosynthetic precursor molecules are used to synthesize a large number of different proteins, polysaccharides & lipids. divergent 55 Summary 56 Electron Transfer Chain Dr. Sugandhika Suresh Dept. of Biochemistry • An overview of cellular respiration High-energy electrons carried by NADH GLYCOLYSIS Glucose Cytoplasmic fluid Figure 6.8 Pyruvic acid KREBS CYCLE ELECTRON TRANSPORT CHAIN AND CHEMIOSMOSIS Mitochondrion During cellular metabolism; Carbohydrates Fat CO2 + H2O Amino acids NADH / FADH2 • Energy stored as NADH / FADH2. • These co-enzymes are further oxidized to free the energy. • If oxidized by a single step „E‟ lost. • A specialized set of electron carriers are used to free the energy and store them in ATP. (electron transfer chain) The Electron Transport Chain • A “specialized set of electron carriers” • Location: inner mitochondrial membrane. • Electrons in the NADH and FADH2 donate electrons to these carriers which pass them down to one another. glycolysis inner membrane outer membrane electron transport chain Krebs cycle H+ e- O2 outer compartment H2O Location inner compartment Of ETC Transport across mitochondrial membrane • NADH produced in glycolysis must be transported to the mitochondrial matrix. MITOCHONDRIAL MEMBRANE TRANSPORT •Adenine nucleotide translocase –Charge across the membrane determines specificity •Phosphate carrier –Proton symport •Di and tri-carboxylic acid carriers –Proton symporters TRANSPORT OF REDUCING EQUIVALENTS OF NADH •Glycerol phosphate shuttle –Two moles of ATP per mole of cytosolic NADH •Aspartate/malate shuttle –Three moles of ATP per mole of NADH • To be an electron carrier the molecules must be able to exist in 2 forms. 1. Oxidized (before accepting electrons) 2. Reduced (after accepting electrons) A 2+ Oxidation A 3+ B 2+ Reduction B+ Redox Pair Oxidation-Reduction potentials of the complexes • In the ETC the electron carriers are organized into groups (multienzyme complexes). • ETC has 5 such complexes. – Complex I, - NADH dehydrogenase – Complex II, - Succinate dehydrogenase – Complex III – Cytochrome reductase – Complex IV- Cytochrome oxidase – Complex V – ATP synthase Each complex has a series of electron carriers Sequence of electron carriers in the respiratory chain Complex I proton pump Coenzyme Q electron shuttle Complex II, does not pump protons Complex III proton pump Cytochrome c electron shuttle Complex IV proton pump Complex I(NADH Dehydrogenase) Takes H from NADH (if you remember from the TCA cycle, NAD+ takes one hydrogen ion and one hydride ion). The H+ ions go to a molecule of FMN which is closely associated to Complex I, turning it into FMNH2 Coenzyme Q then diffuses over to take the hydrogens from FMNH2, and the complex returns to normal, ready to accept hydrogens from the next NADH. CoQ is also reduced by the electrons obtained from the NADH, enabling it to transfer electrons somewhere else Complex II Instead of FMN , FAD which is tightly bound to the complex, and protein complex does not span the entire membrane in the TCA cycle and the Beta-Oxidation Cycle, FADH2 can be produced from reactions involving a couple of different enzymes (e.g. succinate dehydrogenase, malate dehydrogenase or acyl CoA dehydrogenase). substrate such as malate may come along and donate its hydrogens to FAD, forming FADH2, which then passes its H+ ions on to coenzyme Q. Electrons are also transfered to CoQ, reducing it Cytochromes Complexes III(b) ,C ,IV(a) cytochromes contain a ferric ion (Fe3+) which can be reduced to a ferrous ion (Fe2+). This is achieved by the transfer of electrons, as the electrons picked up by CoQ from the other complexes are passed on. Cytochrome b Complex III Cytochrome C Complex IV, Cytochrome a + a3 Since cytochrome a + a3 has copper atoms bound to it, it is able to facilitate the production of water What is oxidative phosphorylation? • involves phosphorylating ADP (to produce ATP) using oxidation reactions. • Building up a proton gradient across the inner membrane of mitochondria, Chemisosmosis and oxidative phosphorylation • Energy is not synthesized directly. • The „E‟ generated at the complexes I, III and IV are used to pump H+ across the inner mitochondrial membrane, into the inter membranous space. inter-membrane space The [H+] in the space increases. This creates an electrochemical gradient (protein motive force) across the inner mitochondrial membrane. cytosol • H+ re-enters the mitochondrial matrix through the ATP synthase molecule. • This re-entry provides the energy for the ATP synthase to synthesize ATP. • Therefore ATP synthesis and e-transport are coupled. Brown Adipose Tissue (BAT) • The energy generated in ETC is used for thermogenesis (heat generation) rather than ATP generation. • Newborns (high BAT – produces heat) • Hibernating animals • In animals exposed to cold. THERMOGENESIS •Uncoupled mitochondria –Brown fat for heat production •Thermogenin • Uncoupling protein Inhibitors of electron transport • Rotenone (a plant toxin used by Amazonian Indians to poison fish and also an insecticide), amytal (a barbiturate), piericidin A (a structural analog of ubiquinone) inhibit at complex I. • Antimycin A ( a Streptomyces antibiotic) inhibits complex III. • CN-, CO, and N3- inhibit complex IV. Certain poisons interrupt critical events in cellular respiration Rotenone Cyanide, carbon monoxide ELECTRON TRANSPORT CHAIN Figure 6.13 Oligomycin ATP SYNTHASE HOW UNCOUPLERS WORK •Uncouplers are weak acids –Become protonated –Carry protons across the membrane –Dissipate the proton gradient •Ionophores such as valinomycin –Carry ions (K+) across the membrane –Dissipate the membrane potential Summary Enzymes as Biological Catalysts • Enzymes are proteins that increase the rate of reaction by lowering the energy of activation • They catalyze nearly all the chemical reactions taking place in the cells of the body • Enzymes have unique threedimensional shapes that fit the shapes of reactants (substrates) Naming Enzymes • The name of an enzyme identifies the reacting substance - usually ends in –ase • For example, sucrase catalyzes the hydrolysis of sucrose • The name also describes the function of the enzyme • For example, oxidases catalyze oxidation reactions • Sometimes common names are used, particularly for the digestion enzymes such as pepsin and trypsin • Some names describe both the substrate and the function • For example, alcohol dehydrogenase oxides ethanol Classification of Enzymes • Enzymes are classified according to the type of reaction they catalyze: Class Oxidoreductases Transferases Hydrolases Lyases Isomerases Ligases Reactions catalyzed Oxidation-reduction Transfer groups of atoms Hydrolysis Add atoms/remove atoms to/from a double bond Rearrange atoms Use ATP to combine molecules Oxidoreductases, Transferases and Hydrolases Lyases, Isomerases and Ligases Active Site of an Enzyme • The active site is a region within an enzyme that fits the shape of substrate molecules • Amino acid side-chains align to bind the substrate through Hbonding, salt-bridges, hydrophobic interactions, etc. • Products are released when the reaction is complete (they no longer fit well in the active site) Enzyme Specificity • Enzymes have varying degrees of specificity for substrates • Enzymes may recognize and catalyze: - a single substrate - a group of similar substrates - a particular type of bond Lock-and-Key Model • In the lock-and-key model of enzyme action: - the active site has a rigid shape - only substrates with the matching shape can fit - the substrate is a key that fits the lock of the active site • This is an older model, however, and does not work for all enzymes Induced Fit Model • In the induced-fit model of enzyme action: - the active site is flexible, not rigid - the shapes of the enzyme, active site, and substrate adjust to maximumize the fit, which improves catalysis - there is a greater range of substrate specificity • This model is more consistent with a wider range of enzymes Enzyme Catalyzed Reactions • When a substrate (S) fits properly in an active site, an enzyme-substrate (ES) complex is formed: E + S ES • Within the active site of the ES complex, the reaction occurs to convert substrate to product (P): ES E + P • The products are then released, allowing another substrate molecule to bind the enzyme - this cycle can be repeated millions (or even more) times per minute • The overall reaction for the conversion of substrate to product can be written as follows: E + S ES E + P Example of an Enzyme Catalyzed Reaction • The reaction for the sucrase catalyzed hydrolysis of sucrose to glucose and fructose can be written as follows: E + S ES E + P1 + P2 where E = sucrase, S = sucrose, P1 = glucose and P2 = fructose Isoenzymes • Isoenzymes are different forms of an enzyme that catalyze the same reaction in different tissues in the body - they have slight variations in the amino acid sequences of the subunits of their quaternary structure • For example, lactate dehydrogenase (LDH), which converts lactate to pyruvate, consists of five isoenzymes Diagnostic Enzymes • The levels of diagnostic enzymes in the blood can be used to determine the amount of damage in specific tissues Temperature and Enzyme Activity • Enzymes are most active at an optimum temperature (usually 37°C in humans) • They show little activity at low temperatures • Activity is lost at high temperatures as denaturation occurs pH and Enzyme Activity • Enzymes are most active at optimum pH • Amino acids with acidic or basic side-chains have the proper charges when the pH is optimum • Activity is lost at low or high pH as tertiary structure is disrupted Optimum pH for Selected Enzymes • Most enzymes of the body have an optimum pH of about 7.4 • However, in certain organs, enzymes operate at lower and higher optimum pH values Enzyme Concentration and Reaction Rate • The rate of reaction increases as enzyme concentration increases (at constant substrate concentration) • At higher enzyme concentrations, more enzymes are available to catalyze the reaction (more reactions at once) • There is a linear relationship between reaction rate and enzyme concentration (at constant substrate concentration) Substrate Concentration and Reaction Rate • The rate of reaction increases as substrate concentration increases (at constant enzyme concentration) • Maximum activity occurs when the enzyme is saturated (when all enzymes are binding substrate) • The relationship between reaction rate and substrate concentration is exponential, and asymptotes (levels off) when the enzyme is saturated Enzyme Inhibitors • Inhibitors (I) are molecules that cause a loss of enzyme activity • They prevent substrates from fitting into the active site of the enzyme: E + S ES E + P E + I EI no P formed Reversible Inhibitors (Competitive Inhibition) • A reversible inhibitor goes on and off, allowing the enzyme to regain activity when the inhibitor leaves • A competitive inhibitor is reversible and has a structure like the substrate - it competes with the substrate for the active site - its effect is reversed by increasing substrate concentration Example of a Competitive Inhibitor • Malonate is a competitive inhibitor of succinate dehydrogenase - it has a structure that is similar to succinate - inhibition can be reversed by adding succinate Reversible Inhibitors (Noncompetitive Inhibition) • A noncompetitive inhibitor has a structure that is different than that of the substrate - it binds to an allosteric site rather than to the active site - it distorts the shape of the enzyme, which alters the shape of the active site and prevents the binding of the substrate • The effect can not be reversed by adding more Irreversible Inhibitors • An irreversible inhibitor destroys enzyme activity, usually by bonding with side-chain groups in the active site