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1、Chapter 13 Overview on metabolism and Principles of Bioenergeticsby Prof. Zengyi Chang (昌增益教授）昌增益教授）Biochemistry Lecture (Nov. 15, 2012) ATPThe study of Metabolism and bioenergetics is key to understand lifeDefining Metabolism The word “metabolism” means change, or overthrow. Biochemically, it means
2、: the entire highly integrated and regulated network of chemical transformations occurring in a living organism, through which cells grow and reproduce, maintain their structures, and respond to their environment.Sanctorius Sanctorius(1561-1636) The “weighing chair Louis Pasteur(1822-1895) Yeast fer
3、mentationcatalyzed by “ferments”.Friedrich Whler(1800-1882) synthesis of urea Eduard Buchner(1860-1917) Cell-free fermentationHans Krebs(1900-1981) Urea cycle & citric acid cycle Metabolism is arbitrarily divided into two categories: The Ying & Yang of Metabolism CatabolismCatabolism ( (biobiodegrad
4、ation): Reactions involving the breaking down of organic substrates, typically by oxidative breakdown, to provide chemically available energy (e.g. ATP) and/or to generate metabolic intermediates used in subsequent anabolic reactions. AnabolismAnabolism (biosynthesis): The processes that result in t
5、he synthesis of cellular components from precursors of lower molecular weight (often via endergonic reactions and thus being energy consuming). Organisms are classified as autotrophs and heterotrophs based on their metabolic features (energy and carbon) Autotrophs (“self-feeding”) Derive energy from
6、 sunlight or inorganic substances, and using CO2 as sole carbon source to produce complex organic compounds; Including green plants, algae, and certain bacteria; Being the “producers” in the food chain. Heterotrophs (“feeding on others”) Derive energy and carbon from oxidation of organic compounds (
7、made by autotrophs); Including all animals and most bacteria and fungi; Being the “consumers” in the food chain.Metabolism in various living organisms lead to the recycling of carbon, oxygen in the biosphere.Autotrophs & heterophs are interdependent on each other in the biosphere.Metabolism leads to
8、 the cycling of nitrogen in the biosphereThe recycling of matter is driven by the flow of energy in one direction through the biosphere, i.e., being constantly transformed into unusable forms such as heat.General Features of Metabolism Occurs in specific cellular locations as a series of enzyme-cata
9、lyzed pathways. Highly coupled and interconnected (“Every road leads to Rome”). Highly regulated to achieve the best economy (“Balanced supply and demand”). The number of reactions is large, however, the number of types of reactions is relatively small (what happens in animal respiration happens in
10、plant photosynthesis). Well conserved during evolution (“what happens in bacteria happens in human being”).EnergyproductionThe basic road map of central metabolic pathways: occurring in three stagesDegradative & biosynthetic pathways are always distinct: for thermodynamics and regulation reasons.Pol
11、ymersMonomersUltimate degradation(乙酰輔酶乙酰輔酶A)Metabolic pathways could be convergent, divergent or cyclic.Anabolic reactions areAnabolic reactions areHighly divergent!Highly divergent!Catabolic reactions areCatabolic reactions areHighly convergent!Highly convergent!Most biochemical reactions fall into
12、 five general categories Making or breaking of C-C bonds; Intramolecular rearrangements (isomerization and elimination); Occur via free-radical intermediates; Group transfer; Oxidation-reduction. The biochemical reactions have been products of evolutionary selection based on their relevance for the
13、life process, as well as their rates (after being catalyzed by proper enzymes), i.e., not all the organic reactions you learned in organic chemistry occur in living cells.Thousands of biochemical reactions might occur in the human Computational prediction in humans: a Computational prediction in hum
14、ans: a total of 1653 metabolic enzymes, only 622 total of 1653 metabolic enzymes, only 622 of which were assigned roles in 135 of which were assigned roles in 135 predicted metabolic pathways. predicted metabolic pathways. Uncharacterized pathways?Uncharacterized pathways?References:References:1.Rom
15、ero et al., (2004) “Computational prediction of human metabolic pathways from the complete human genome”, Genome Biology, 6:R2. 2. Smith, E. and Morowitzj, H. J. (2004) “ Universality in intermediary metabolism”, PNAS, 101: 1316813173.3. Caetano-Anolles, G., Kim, H. S. and Mittenthal, J. E. (2007) “
16、The origin of modern metabolic networks inferred from phylogenomic analysis of protein architecture. PNAS, 104: 935863. Issues for current and future investigation on metabolism Observation of metabolic processes in intact living organisms (e.g., in the brains under various states) Continue to unvei
17、l new pathways and new regulation strategies of metabolism. Studies on enzymes. Metabolism differences among various organisms or various states of the same organism (for diagnosing and treating such diseases as cancer, infections of bacteria or viruses, obesity, etc; to understand aging). Appropria
18、te and inappropriate nutrition. Biotechnological application of knowledge learned from metabolic studies in medicine, agriculture and industry. How one should learn about metabolism Compare and relate (interconnect) the chemical reactions (Where are you in the metabolism network?) Try to contemplate
19、 on the ways the living organisms used to achieve a balanced and dynamic steady state (How could the multilayered regulation cooperate so effectively?). Understand the classical experiments and thoughts that led to the revelation of the knowledge described (Why he/she won the Nobel Prize?). Be aware
20、 of the nature of the data (Could the in vitro observations be extended to what happens in vivo?). Understand the aspects that need further studies (How could I win a Nobel Prize?).Bioenergetics The quantitative study (mainly using the principles of chemical thermodynamics) of energy transductions i
21、n living cells and the physical-chemical nature underlying these processes.Bioenergetics began with early quantitative studies on animal respiration Lavoisier used a calorimeter to estimate heat produced (water melted from ice) by the guinea pigs metabolism: Animal respiration (transport of O2 from
22、air to tissues, and CO2 in opposite direction) is nothing but slow combustion of carbon and hydrogen, like that of a candle burning (1789). Key issues: Where is O2 converted to CO2 and H2O in animals? What contributes the carbon and hydrogen? A. Lavoisier(1743-1794)Measuringheat produced;O2 taken in
23、;H2O produced;CO2 produced. Using guinea pig.A scientific understanding of animal respiration (biological oxidation) Location of biological oxidation: Lung blood all tissues all cells Revelations of the molecular mechanism: How O2 participates (production of CO2 and H2O); What enzymes (cytochromes,
24、dehydrogenases, etc) participate; What are the roles of iron and light absorbing redox components (heme groups); What subcellular locations (mitochondria); How to study quantitatively for the energy transformation (a thermodynamics approach); etc. The Energy concept was established by physicists (19
25、th century) An abstract numerical physical quantity (indirectly observed) or mathematical principle that indicate the ability of a system to do work. It is not a description of a mechanism or anything concrete. Energy can neither be created nor destroyed: It can only be transformed from one form to
26、another (Helmholtz, 1847; proposed in the context of his studies on muscle metabolism). The Gibbs free energy-the energy that can be converted into work at a uniform temperature and pressure throughout a system (Gibbs, 1876). J. P. Joule(1818-1889) H. von Helmholtz(1821-1894) J.W.Gibbs(1839-1903)The
27、 Gibbs free energy concept was applied to study chemical reactions Gibbs developed the chemical thermodynamics: relating free energy change with equilibrium constant. G G = = G G o o + + RT RT ln Qln Q (Q = products/reactants) G G o o = - = -RT RT ln ln K K eqeq (K Keqeq : equilibrium constant) The
28、actual free energy change (The actual free energy change ( G G ) ) determines whether a reaction occurs favorably (or spontaneously). The standard free energy changeThe standard free energy change in biochemistry ( Go) is a constant (measured under a standard set of conditions).G for a reaction can
29、be larger, smaller, or the same as Go, depending on the concentrations of the reactants and products (Q: mass action ratio).J.W.Gibbs(1839-1903) A small change in standard free energy leads to a large change in equilibrium constantLiving organisms have to consume energyTo generate and maintain its h
30、ighly ordered structure (biosynthesis).To generate motion (mechanical work).To generate concentration and electrical gradients across cell membranes (active transport).To generate heat and light in certain organisms.The “energy industry” (production, storage and The “energy industry” (production, st
31、orage and use) is central to the economy of the cell use) is central to the economy of the cell society!society! Living organisms consume free energy Living cells are generally held at constant temperature and pressure: chemical energy (Gibbs free energy, G- “available energy”) has to be used by liv
32、ing organisms.Living organisms require a continual input of free energy.Biological energy transformation obey the two basic laws of thermodynamics.Free energy change in oxidation-reduction reactions can be calculated by measuring the reduction potential Reduction potential (in volts or millivolts) m
33、easures the tendency of a chemical species to acquire electrons and thereby be reduced. The more positive the potential, the greater the species affinity for electrons and tendency to be reduced. Standard reduction potential (Eo) isdefined relative to a reference electrode.e-e-E o = 0.00 VE o = 0.00
34、 VpH 7Negative E o pH 7positive E o pH 0pH 0Standard reduction potentials of biologically important half-reactions have been systematically measured. The actual reduction potential (E) of each half-reaction can be calculated according to the Nernst equation The actual reduction potential (E) depends
35、 on the standard reduction potential (Eo ), electrons transferred per molecule (n), temperature (T), ratio of reduced form/oxidized form:reduced formoxidized formEEoWalther Nernst(1864-1941) G of a redox reaction can be directly calculated from the value of E (= E of the electron acceptor E of the e
36、lectron donor): G can be calculated from E using the Nerst EquationWhen E is positive, G is negative.The thermodynamics concepts applied in biochemical studies (since 1930s) Borsook and Schott (1931) The role of the enzyme in the succinate-enzyme-fumarate equilibrium, J. Biol. Chem. 92:535-557. Bors
37、ook, H.& Schott, H. F. (1931) The free energy, heat, and entropy of formation of l-malic acid. J. Biol. Chem. 92:559-567. Reduction potentials,equilibrium constants,heat capacities measured,free energy, entropy calculated.The thermodynamic concepts were applied in studying the synthesis of proteins
38、(1930s)Borsook and Huffman, (1938). Some thermodynamical considerations of amino acids, peptides, and related substances, in Chemistry of the Amino Acids and Proteins (C. L. A. Schmist, editor) C. C. Thomas, Springfield, Ill.It was originally thought the synthesis should occur by Mass Action in reve
39、rsal, as catalyzed by proteases, glycogen phosphorylases, and polynucleotide phosphorylases. It was later realized that the free energy change in hydrolysis was so large that one could not get synthesis by any feasible degree of concentration of amino acids. Energy is needed to be put into the syste
40、m, via a coupled reaction. Amount of free energy expended to form the peptide bonds or phosphodiester bonds in vivo is far higher than their standard free energy of formation in vitro, to buy specificity of the bonds formed! A direct source of energy for muscle contraction was searched!Non-lactic mu
41、scle contractions at the expense of the dephosphorylation of creatine phosphate (1930) Lundsgaard, E., Biochem. Z. 217, 162; 227, 51 (1930). Frog muscles poisoned with iodoacetate (unable to split glucose to lactic acid) are capable of carrying out contractions！ A parallel breakdown of creatine phos
42、phate observed in the presence of iodoacetate. LundsgaardLactic acid does not serve asan energy source for muscle contraction!Creatine phosphateA rapidly mobilizable reserve of high-energy phosphates in skeletal muscle and the brain.ATP discovered in muscle (1929)Lohmann, (1928) Ueder das Workommen
43、und Umsatz von Pyrophosphat in der Zelle, Biochem. Z, 202:466-493; 203:164-207.Fiske and Subbarow (1929) Phosphorus Compounds of Muscle and Liver, Science, 70:381 382.Langen & Hucho (2008) Karl Lohmann and the Discovery of ATP, Angewandte Chemie, 47:1824-1827. From Fiske and Subbarow paper.Myosin wa
44、s found to be an ATPase Engelhardt WA, Liubimova MN. (1939) Myosine and adenosinetriphosphatase, Nature, 144:688 Acidification to pH below 4 rapidly destroys this activity; completely lost after 10 min at 37oC, but the present of ATP stabilizes it.Engelhardt (1941):The free energy coupling and ATP e
45、nergy currency theories proposed (1941)Lipmann, F. (1941). “Metabolic Generation and Utilization of Phosphate Bond Energy”. Advances in Enzymology and Related Subjects, 1:99-162.Kalckar, H, (1941). “The Nature of Energetic Coupling in Biological Synthesis” Chem. Rev. 28:71-178. “Energy-rich phosphat
46、e” (like ATP) proposed to drive energy-requiring biological processes (e.g, Muscle contraction, transport of ions and other molecules across membranes, chemical reaction for the biosynthesis of proteins and nucleic acids). The biological oxidoreduction (respiration) is compulsorily coupled to phosph
47、orylation. LipmannKalckarATP is the universal currency for biological energy This was first perceived by Fritz Lipmann and Herman Kalckar in 1941 when studying glycolysis. Hydrolysis of the two phosphoanhydride (磷酸酐鍵) bonds in ATP generate more stable products releasing large amount of free energy (
48、Go is -30.5 kJ/mol; G Gp p in in cells is -50 to -65 kJ/molcells is -50 to -65 kJ/mol). The ATP molecule is kinetically stable at pH 7 and enzyme catalysis is needed for its hydrolysis. ATP actually exists as a sum of various species in cells (e.g., ATP4- and MgATP2-).Fritz Lipmann(1899-1986) Fig. 2
49、. The two-dimensional stick model of the adenosine phosphate family of molecules, showing the atom and bond arrangement.The human body on average contains only 250 grams of ATP but turns over its own body weight equivalent in ATP each day!ATP provides ATP provides energy usually energy usually throu
50、gh groupthrough grouptransfer transfer (protein could(protein couldalso be suchalso be suchacceptors)acceptors)Gln synthetaseKeq = 10 - Go/1.36 The reaction is thus accelerated by about 105 fold!ATP might be considered as a “coenzyme” in this sense.Concept of coupled reactions formulated (1900) Wilh
51、elm Ostwald, (1900) Z. Physik. Chem. 34:248 The free energy released by an exergonic process can be used to drive an endergonic process that would not go by itself. “Waste no (free) energy; use it well.Nucleophilic attacksNot phosphateATP usually provides energy by group transfer of phosphoryl group
52、s (磷?；柞；? -PO32-), not phosphate groups (-OPO32-), forming covalent intermediates, not by simple hydrolysis.For energy to be supplied, the two processes have to be coupled!ATP supplies energy for all kinds of cellular processesATP has an intermediate phosphoryl group transfer potential, thus ADP ca
53、n accept and ATP can donate phosphoryl groups (forming the ATP-ADP cycle and acting as an energy currency)Why does ATP have a high phosphoryl transfer potential? G0 depends on the difference in free energies of products and reactants, therefore, both must be considered ; Thereis no such thing as “hi
54、gh energy bond”.Four factors are important:1. Relief of charge repulsion;2. Resonance stabilization;3. Ionization;4. Stabilization due to hydration.ATP is not a long-term storage form of free energy in living cells, but phosphocreatine is one such phosphoryl reservoir, or so-called phosphagen (also
55、inorganic polyphosphate).Biological energy was found to be produced via oxidation-reduction reactions (i.e., electron transferring) Metabolic fuelsCO22e- 2e-2e-2e-H2OO2ATPFree energyOxidationMetabolic fuels are oxidized to CO2, with electrons transferred first to universal carriers (e.g., NAD+ and F
56、AD), and eventually to O2.Energy is released during such redox reactions and eventually conserved in ATP.(NADH, FADH2)TransmembraneProton gradientFunctional groups in organic compounds present in one of four general oxidation states, equivalent to alkane, alcohol, ketone, or carboxylic acidIn aerobi
57、c organisms, the ultimate electron acceptor in theoxidation of carbon is O2, and the oxidation product is CO2Nicotinamide adenine dinucleotide (NAD+) was found to be a common cofactor for hydrogen-transferring enzymes (1906) Harden & Young (1906). The Alcoholic Ferment of Yeast-Juice. Proc.Roy.Soc.7
58、8: 369375. Warburg & Christian (1936). Pyridin, the hydrogen-transferring component of the fermentation enzymes (pyridine nucleotide). Biochemische Zeitschrift 287: 291-328. Being thermostable and filtratable;Composed of adenine, nicotinamide,pentose, and phosphate in the ratio1:1:2:3.The nicotinami
59、de portion reversiblycarries hydrogen. HardenWarburgNAD/NADP found to be universal electron carriersAbsorbs light at 340 nm upon reduction.NADP+: Nicotinamide adenine dinucleotide phosphate.hydride ion(two electrons)Being soluble coenzymes of dehydrogenasesFMN/FAD was found as a common iron-free yel
60、low pigment involved in respiration (1934) Ellinger and Koschara (1934) The lyochromes: A new group of animal pigments. Nature 133:553-556. Flavin adenine dinucleotide (FAD) Flavin mononucleotide (FMN), FAD/FMN also found to be universal electron carriers (in flavoproteins)Being stronger oxidizing a
61、gents than NAD/NADP and are particularly useful because they can take part in both one- and two-electron transfers. Often tightly bound to dehydrogenases (thus being their prosthetic groups)FAD (FMN)(Oxidized) The vitamins niacin (nicotinic acid) and riboflavin (vitamin B2) provide precursors for ma
62、king NAD/NADP and FAD/FMN Riboflavin (vitamin B2)NAD+FADNiacinAdenosine diphosphate (ADP) is an ancient module in metabolismEvolved from early RNA catalysts?Both NAD+ and FAD are derivatives of ATP!Summary Bioenergy is chemical energy, studied in terms of free energy and free energy change (G ). ATP
63、 acts as the free energy carrier (currency) in cells. Bioenergy is mainly produced via stepwise electron flow (redox reactions) through a series of electron carriers having increasing levels of reduction potential (E). Electrons released from the oxidation of nutrient fuels are initially channeled to a few universal electron carriers (mainly NADH and FADH2).
生物化學(xué)：chapter 13 Overview of Metabolism and Principles of Bioenergetics
