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Chapter 15 Metabolism: Basic Concepts and Design The tiny ruby-throated hummingbird can store enough fuel and converts fuels into the cellular energy currency, ATP. Fly 500 miles across Gulf of Mexico without resting! 1 Outline 15.1 Metabolism is composed of many coupled, interconnecting reactions 15.2 ATP Is the universal currency of free energy in biological systems 15.3 The oxidation of carbon fuels is an important source of cellular energy 15.4 Metabolic pathways contain many recurring motifs 2 All Cells Transform Energy : • Cells extract energy from their environment and use this energy to convert simple molecules into cellular components Metabolism : • A highly integrated network of chemical pathways that enables a cell to extract energy from the environment and use this energy for biosynthetic purposes. 3 Metabolism answers the questions • How does a cell extract energy and reducing power from its environment? • How does a cell synthesize the building blocks of its macromolecules and then the macromolecules themselves ? 4 General Principles and motifs of metabolism 1. Metabolic pathways : step by step 2. Common energy currency: ATP, links energyreleasing pathways with energy-requiring pathways 3. Oxidation of carbon fuels powers the formation of ATP 4. Unifying themes : a limited number of types of reactions and particular intermediates are common to many pathways 5. Metabolic pathways are highly regulated 5 15.1 Metabolism Is Composed of Many Coupled, Interconnecting Reactions • Why Do Organisms Need Energy? – Performance of mechanical work in muscle contraction and cellular movements – Active transport of molecules and ions – Synthesis of macromolecules and other biomolecules from simple precusors 6 15.1 Metabolism Is Composed of Many Coupled, Interconnecting Reactions •Phototrophs or photosynthetic organisms (光合有機體): – Organisms that obtain energy by trapping sunlight and convert light energy into chemical energy • Chemotrophs (化能生物) – Organisms that obtain energy by the oxidation of foodstuffs generated by phototrophs 7 Metabolism consists of energy-yielding and energy-requiring reactions •Metabolism –a linked series of chemical reactions that begins with a particular molecule and converts it into some other molecule or molecules in a carefully defined fashion –Many such defined pathways in the cell •These pathways are interdependent •Their activity is coordinated by allosteric enzyme 8 無氧 有氧 CO2 Fig 15.1 Glucose metabolism 9 Fig 15.1 Metabolic pathways 10 • Metabolic pathways: – Catabolism (分解代謝) or catabolic reaction • Convert energy from fuels into cellular energy – Anabolism (合成代謝) or anabolic reactions • Require energy to synthesizes molecules from simpler precursors catabolism Fuel (carbohydrate, fats) CO2 + H2O + useful energy Useful energy + simple precursor anabolism Complex molecules Amphibolic pathways: some pathways can be either anabolic or catabolic, depending on the energy conditions in the cells General principle of metabolism is that biosynthetic and degradative pathways are almost always distinct 11 Catabolism and Anabolism 氧化、放熱 還原、吸熱 NADH Fig. 17.6 in Garrett and Grisham, Biochemistry, 4th edition (2009) 12 A Thermodynamically Unfavorable Reaction Can Be Driven by a Favorable Reaction • A pathway must satisfy minimally two criteria: (1) Individual reactions must be specific • Yield only one particular product or sets of products (2) Entire set of reactions that constitute the pathway must be thermodynamically favored • Free energy (ΔG) is negative 13 Free-energy change: Spontaneity (自發性) but not the rate of reaction ΔG tells us if the reaction can occur spontaneously: 1. If G is negative, reaction spontaneous, exergonic 2. If G is zero, no net change, system at equilibrium 3. If G is positive, free energy input required, endergonic G of a reaction depends only on free-energy of products minus free-energy of reactants 1. G of a reaction is independent of path (or molecular mechanism) of the transformation 2. G provides no information about the rate of a reaction ----The rate of a reaction depends on the free energy of activation (G‡ 活化能), which is largely unrelated to the G of the reaction---- 14 15 Standard Free-energy change of a reaction is related to the equilibrium (化學反應標準自由能變化與平衡常數有關, Go’ of a reaction is related to K’eq) •To determine G of an enzyme catalyzed reaction , must consider nature of both reactants and products as well as their concentrations Consider this reaction A+BC+D G is given by G = Go + RTln([C][D]/[A][B]) (1) G change in free energy Go is standard free-energy change (Go’ : Go at pH7) R is the gas constant, T is the absolute temperature [C][D]/[A][B] are the molar concentrations of the reactants G of a reaction depends on the nature of the reactants 16 (Go ) and on their concentrations For biochemical reactions: • At equilibrium, G = 0 and so 0 = Go’ + RTln([C][D]/[A][B]) Go’ = - RTln([C][D]/[A][B]) (2) (3) •Equilibrium constant under standard conditions, K’eq, is defined as K’eq = [C][D]/[A][B] (4) Substituting equation 4 into equation 3 gives Go’ = - RTlnK’eq K’eq =10 -Go’/RT (5) (6) R = 1.987x10-3 kcal mol-1 deg-1 and T = 298K (=250C) K’eq =10 -Go’/2.47 (7) 17 A Thermodynamically Unfavorable Reaction Can Be Driven by a Favorable Reaction • Free energy changes are additive: Overall ΔG for a series of reactions is equal to the sum of ΔG of each individual reaction A B + C ΔG o’ = +21kJ/mol BD ΔG o’ = -34kJ/mol A C + D ΔG o’ = -13kJ/mol A thermodynamically unfavorable reaction can be driven by a thermodynamically favorable reaction to which it is coupled 18 15.2 ATP Is the universal currency of free energy in biological system ATP (adenosine triphosphate) – Acts as the free-energy donor in most energy-requiring process • Motion • Active transport • Biosynthesis • Most of catabolism consists of reactions that extract from fuels and convert it into ATP 19 ATP structure •ATP is a nucleotide consisting of an adenine, a ribose, and a triphosphate unit •The active form of ATP is usually a complex of ATP with Mg2+ or Mn2+ •ATP is an energy-rich molecule –its triphosphate unit contains two phosphoanhydride bonds –ATP hydrolysis is exergonic (ΔG < 0) •ATP + H2O ADP + Pi ΔGo’ = -30.5kJ/mol •ATP + H2O AMP + Pii ΔGo’ = -45.6kJ/mol triphosphate unit Anhydride bond adenine The precise ΔGo’ of ATP hydrolysis depends on: • Ionic strength of the medium • The concentration of Mg2+ or other metal ions [ MgADP ] [ Pi ] 20 G G ' RT ln 2 [ MgATP ] 0 Actual ΔG of ATP hydrolysis in human erythrocyte •pH 7, temperature 37 oC **ATP is formed from ADP and Pi ATP-ADP cycle The fundamental mode of energy exchange in biological systems 21 Active form of ATP • A complex of ATP with Mg2+ or Mn2 + – True substrate for the enzyme 22 The role of ATP in energy metabolism is paramount • Some biosynthetic reactions are drive from GTP, UTP or CTP – Nucleoside monophosphate kinase catalyzed the phosphorylation – All the nucleotide triphosphates are energetically equivalent • NAD+ and FAD (electron carriers) are derivatives of ATP NMP Nucleoside monophosphate NDP Nucleoside diphosphate Nucleoside monophosphate kinase + ATP NDP Nucleoside diphosphate kinase + ATP NTP + ADP + ADP 23 ATP Hydrolysis Drives Metabolism by Shifting the Equilibrium of Coupled Reactions •A thermodynamically unfavorable reaction can be driven by a thermodynamically favorable reaction to which it is coupled reaction 24 ATP Hydrolysis Drives Metabolism by Shifting the Equilibrium of Coupled Reactions – Coupling the hydrolysis of ATP with the conversion of A into B under standard conditions has changed equilibrium ratio of B to A by a factor of about 105. – If used the energy of ATP hydrolysis under cellular conditions, ratio of [B] to [A] changes by about 108 (if using -50.2 kJ mol-1). –ATP’s action as an energy-coupling agent 25 A + ATP + H2O B + ADP + Pi •The hydrolysis of n ATP molecules changes the equilibrium ratio of a couple reaction by a factor of 108n •A thermodynamically unfavorable reaction sequence can be converted into a favorable one by coupling it to the hydrolysis of a sufficient number of ATP molecules in a new reaction •A and B may represent activated and unactivated conformations of a protein • Activated by phosphorylation with ATP • Such as: Muscle contraction •A and B may refer to the concentrations of an ion or molecule on the outside and inside of a cell • Active transport of Na+ and K+ across membrane 26 ATP has a higher phosphoryl transfer potential (phosphoryl-group transfer potential) ATP + H2O ADP + Pi + H+ ∆Go’=-30kJ mol-1 (-7.3kcalmol-1) Glycerol 3-phosphate + H2O glycerol + Pi ∆Go’=-9.2kJ mol-1 (-2.2kcalmol-1) •ATP has a stronger tendency to transfer its terminal phosphoryl group to water than does glycerol 3phosphate •ATP has a higher phosphoryl transfer potential (phosphoryl-group transfer potential) than does glycerol 3-phosphate. 27 Higher phosphoryl transfer potential of ATP due to ATP structure 1. Resonance Stabilization – ADP and Pi, have greater resonance stabilization than does ATP 2.Electrostatic repulsion : – At physiological pH 7 , charge on ATP is -4 – Strong electrostatic repulsion between them – Repulsion is reduced when ATP is hydrolyzed 3.Stabilization due to hydration: – More water can surround ADP and Pi than ATP, increasing stabilization by hydration Fig 15.5 Improbable resonance structure : 28 Fig 15.4 Resonance structures of orthophosphate terminal part of ATP- γ phosphate group ATP is not the only compound with a high phosphoryl-transfer potential 29 Creatine Phosphate • Creatine phosphate in vertebrate muscle serves as a reservoir of high-potential phosphoryl groups that can be readily transferred to ATP Creatine kinase Creatine phosphate + ADP ATP + creatine ΔG°’ = -12.6 kJ/mol Keq = 162 30 Sources of ATP during exercise Fig 15.7 Source of ATP during exercise •ATP stored in muscle can sustain activity for < 1 sec •Creatine phosphate also stored in muscle – major source of ATP regeneration for next several seconds • Initially exercise is powered by existing ATP and creatine phosphate •Subsequently the ATP must be generated by metabolic pathways 31 15.3 The oxidation of carbon fuels is an important source of cellular energy •ATP is an immediate donor of free energy, not a long-term storage form of free energy (ATP提供能量但非儲存能量分子) –ATP is consumed within ~1 min of its formation (Total ATP ~100 g –turnover is very high •In 24 hours, a resting human turns over 40 kg ATP •During exercise, turnover up to 0.5 kg ATP/min •Regenerating ATP –Oxidation of fuel molecule –Electrons are used to regenerate from ADP and Pi. Fig 15.18 ATP-ADP cycle 32 General Principles and motifs of metabolism 1. Metabolic pathways : step by step 2. Common energy currency: ATP, links energy-releasing pathways with energy-requiring pathways 3. Oxidation of carbon fuels powers the formation of ATP 4. Unifying themes : a limited number of types of reactions and particular intermediates are common to many pathways 5. Metabolic pathways are highly regulated 33 Free Energy of Oxidation of Single-Carbon Compounds •In aerobic organisms, the ultimate electron acceptor in the oxidation of carbon is O2 and the oxidation product is CO2 •The more reduced a carbon is to begin with, the more free energy is release by its oxidation O 陰電性 > C陰電性 C 陰電性 > H陰電性 most oxidized most reduced e- 8 6 4 2 0 •Oxidation: loss of electrons Fig 15.9 free energy of oxidation of 34 Reduction: gain of electrons single-carbon compounds •Fats are a more efficient fuel source than carbohydrates such as glucose because the carbon in fats is more reduced –A fuel molecules: oxidation takes place one carbon at a time –Fatty acid is more reduced than glucose and can produce more energy •Carbon oxidation energy is used to create –Compounds with high phosphoryl-transfer potential –Ion gradient •End point is formation of ATP Fig 15.10 Prominent fuels 35 High Phosphoryl transfer potential compounds can couple carbon oxidation to ATP synthesis •Energy released in the oxidation of a carbon compounds is converted into ATP •Example GAP (G-3-P) a metabolite of glucose aldehyde acid + energy (GAP) -however the oxidation does not take place directly 36 high phosphoryl transfer potential •The carbon oxidation generates an acyl phosphate, 1,3bisphosphoglycerate •The electron released are captured by NAD+. 37 Compounds with high phosphoryl-transfer potential can couple carbon oxidation to ATP synthesis • The cleavage of 1,3-BPG can be coupled to synthesis of ATP • The energy of oxidation is initially trapped as a high-energy phosphate compound (1,3-bisphosphoglycerate) and then used to form ATP 38 Ion gradients across membranes provide an important form of cellular energy that can be coupled to ATP synthesis •Electrochemical potential of ion gradients across membranes, produced by the oxidation of fuel molecule or by photo-synthesis, is the most common way to form ATP •In animals, proton gradients account for > 90% ATP generation, called oxidative phosphorylation Fig 15.11 Proton gradient 39 Energy from foodstuffs is extracted in three stage •First stage (preparation) – Large molecules in food are broken down into smaller units digestion – Absorbed by intestine cells and throughout the body – No useful energy is captured •Second stage – The small molecules are degraded to simple units that play a central role in metabolism – Convert into acetyl CoA – Some ATP is generated •Third stage – ATP is produced from the complete oxidation of acetyl CoA – Citric acid cycle – Oxidative phosphorylation Fig 15.12 Stages of catabolism gradient40 • In stage III – Acetyl CoA brings acetyl units into the citric acid cycle – then oxidized to CO2 • 3 e- pairs transferred to NAD+, 1 e- pair transferred to FAD for each acetyl group – proton gradient is generated as electron flow from NADH and FADH2 to O2, gradient used to make ATP (oxidative phosphorylation) Generating high-energy electrons Converting the energy of these electrons into ATP 41 General Principles and motifs of metabolism 1. Metabolic pathways : step by step 2. Common energy currency: ATP, links energy-releasing pathways with energy-requiring pathways 3. Oxidation of carbon fuels powers the formation of ATP 4. Unifying themes : a limited number of types of reactions and particular intermediates are common to many pathways 5. Metabolic pathways are highly regulated 42 15.4 Metabolic pathways contain many recurring motifs Activated Carriers •Activated carrier of phosphoryl groups –ATP (because phosphoryl transfer from ATP is an exergonic process) •Activated carriers of electrons for fuel oxidation •Nicotinamide adenine dinucleotide (NAD+) •Flavin adenine dinucleotide (FAD) •Activated carriers for reductive biosynthesis : NADPH •Activated carriers of two-carbon fragments: Coenzyme A 43 Many Enzyme require Cofactors •A complete, catalytically active enzyme together with its bound coenzyme and/or metal ions is called a holoenzyme Apoenzyme + cofactor = holoenzyme •An enzyme without its cofactor is referred to as an apoenzyme. •Cofactors can be subdivided into two groups: (1) small organic molecules called coenzyme (2) metals •Tightly bound coenzyme are called prosthetic groups; loosely associated coenzymes are cosubstrates. •Enzymes that use the same coenzyme usually perform catalysis by similar mechanisms :vitamins 44 Activated carriers of electrons for fuel oxidation •In aerobic organisms, the ultimate electron acceptor in the oxidation of fuel molecules is O2 –fuel molecules transfer electrons to special carriers, which are either NADH or FADH2 (intermediate carriers) –And then transfer electron to O2 45 Activated carriers of electrons for fuel oxidation •NADH (nicotinamide adenine dinucleotide) –Reactive part: nicotinamide ring •pyridine derivative synthesis from the vitamin niacin (維他命B3, 菸鹼酸) •Accepts a hydrogen ion and two electrons R = H for NAD+ adenine R = PO32- for NADP+ Fig 15.13 Structures of the oxidized forms of nicotinamide-derived electron carriers oxidized form Dehydrogenation Dehydrogenase Reduced form 46 NAD+ and NADP+ (a) Nicotinamide adenine dinucleotide, NAD+, and its phosphorylated analog NADP+ undergo reduction to NADH and NADPH, accepting a hydride ion (two electrons and one proton) from an oxidizable substrate. The hydride ion is added to either the front (the A side) or the back (the B side) of the planar nicotinamide ring. (b) The UV absorption spectra of NAD+ and NADH. Reduction of the nicotinamide ring produces a new, broad absorption band with a maximum at 340 nm. The production of NADH during an enzymecatalyzed reaction can be conveniently followed by observing the appearance of the47 absorbance at 340 nm. • Flavin Adenine Dinucleotide (FAD) – consists of a flavin mononucleotide (FMN) unit and an AMP unit – Reactive part: isoalloxazine ring • A derivative of vitamin riboflavin (維生素B2,核黃素) • Accepts two electrons and two protons oxidized form Reduced form adenine Fig 15.14 Structures of the oxidized forms of flavin adenine dinucleotide (FAD) Fig 15.15 Structures of the reactive parts of FAD and FADH2 48 Activated carriers for reductive biosynthesis: NADPH •High-potential electrons are required in most biosyntheses because the precursors are more oxidized than the products •Electron donor in most reductive biosyntheses is NADPH (nicotinamide adenine dinucleotide phosphate) –2’-hydroxyl group of adenosine moiety is esterified with phosphate –is used almost exclusively for reductive biosyntheses (NADH is used primarily for the generation of ATP) •NADPH provides electrons 49 •NADPH operates chiefly with enzymes that catalyze anabolic reactions, supplying the high-energy electrons needed to synthesize energy-rich biological molecules •NADH, as an intermediate in the catabolic system of reactions that generate ATP through the oxidation of food molecules •The genesis of NADH from NAD+ and that of NADPH from NADP+ occurs by different pathways that are independently regulated, so that the cell can adjust the supply of electrons for these two contrasting purposes •Inside the cell, the ratio of NAD+ to NADH is kept high, whereas the ratio of NADP+ to NADPH is kept low. This arrangement provides plenty of NAD+ to act as an oxidizing agent and plenty of NADPH to act as a reducing agent—as required for their special roles in catabolism and anabolism 50 Essential Cell Biology by Alberts, Bray, Hopkin, Johnson, Lewis, Raff, Roberts, and Walter, 4th edition published by Garland Science Activated carriers of two-carbon fragments: Coenzyme A •Coenzyme A (CoA-SH) –a carrier of acyl groups derived from the vitamin pantothenate (維生素B5,泛酸) •The terminal sulfhydryl group is the reactive site •Acyl groups are important constituents both in catabolism and in anabolism – Acyl groups are linked to CoA by thioester bonds – An acyl group often linked to CoA is the acetyl unit acetyl CoA – Acetyl CoA carries an activated acetyl group, just as ATP carries an activated phosphoryl group. thioester bonds 51 52 Acetyl CoA has a high acetyl grouptransfer potential •The ΔGo’ for the hydrolysis of acetyl CoA has a large negative value •The hydrolysis of a thioester is thermodynamically more favorable than that of an oxygen ester Acetyl CoA + H2O acetate + CoA + H+ ΔGo’ = -7.5kcal mol-1(-31.4kJ mol-1) oxygen ester thioester •Acetyl CoA carries an activated acetyl group •Transfer of the acetyl group is exergonic •Acetyl CoA has a high acetyl 53 group-transfer potential •Use of activated carriers illustrates two key aspects of metabolism: –NADH, NADPH, and FADH2 react slowly with O2 in the absence of a catalyst kinetic stability it enables enzymes to control the flow of free energy and reducing power –Most interchanges of activated groups in metabolism are accomplished by a rather small set of carriers •The existence of a recurring set of activated carriers in all organisms is one of the unifying motifs of biochemistry 54 Important!!!! 55 Many activated carriers derived from vitamins •Almost all of the activated carriers that act as coenzymes are derived from vitamins – Vitamins are organic molecules that are needed in small amounts in the diets of some higher animals 腳氣病 口角幹裂、皮膚炎 糙皮病 56 Structures of some of the B vitamins :泛酸 CoA FAD :核黃素 NAD+ :菸鹼酸 57 • Humans beings require at least 12 vitamins in their diet • Not all vitamins coenzymes, e.g., A, C, D, E, K 58 59 General Principles and motifs of metabolism 1. Metabolic pathways : step by step 2. Common energy currency: ATP, links energy-releasing pathways with energy-requiring pathways 3. Oxidation of carbon fuels powers the formation of ATP 4. Unifying themes : a limited number of types of reactions and particular intermediates are common to many pathways 5. Metabolic pathways are highly regulated 60 Thousands of metabolic reactions can be subdivided into just six types 氧化還原酶 轉移酶 水解酶 裂解酶 異構酶 連接酶 電子或質子轉移 官能基團的轉移 加水或脫水分子 共價鍵生成或裂解 同一分子內基團之轉移 消耗 ATP 生成分子 間新鍵 61 • Oxidation-reduction reactions – Electron transfer – Such as: oxidoreductase or dehydrogenase – Useful energy is often derived from the oxidation of carbon compounds – FADH2 and NADH are electron carrier Succinate dehydrogenase Malate dehydrogenase 62 •Ligation reactions –Require ATP cleavage –Formation of covalent bonds (C-C bonds) –Combine smaller molecules to form larger ones pyruvate carboxylase 63 •Isomerization Reactions –Rearrangement of atoms to form isomers –Prepare the molecules for subsequent reactions Aconitase 64 •Group-transfer reactions –Transfer of a functional group from one molecule to another –Such as: phosphoryl group hexokinase 65 •Hydrolytic Reactions –Cleavage of bonds by the addition of waters –Break down large molecules to facilitate further metabolism or to reuse some of the components for biosynthesis purposes –Hydrolysis of a peptide to yield two smaller peptides 66 • Functional groups may be added to double bonds to form singles bonds or removed from single bonds to form double bonds – Addition or removal of groups to form double bonds – Such as: lyases Aldolase Enolase 67 General Principles and motifs of metabolism 1. Metabolic pathways : step by step 2. Common energy currency: ATP, links energy-releasing pathways with energy-requiring pathways 3. Oxidation of carbon fuels powers the formation of ATP 4. Unifying themes : a limited number of types of reactions and particular intermediates are common to many pathways 5. Metabolic pathways are highly regulated 68 Metabolism is regulated in three principal ways •Controlling the amounts of enzymes –The amount of a particular enzyme depends on both its rate of synthesis and degradation –Adjusted by the transcription rate •Presence of Lactose in E. coli induces synthesis of βgalactosidase •Controlling catalytic activities •Controlling accessibility of substrates 69 • Controlling catalytic activities – Reversible allosteric control • First reaction in biosynthetic pathways is allosterically inhibited by end product of the pathway • Feedback inhibition – Inhibition of aspartate transcarbamoylase by cytidine triphosphate – Reversible covalent modification • triggered by hormones and signal transduction. – Glycogen phosphorylase is activated by serine phosphorylation when glucose is scarce. – Hormone coordinate metabolic relations between different tissue – Energy status of the cell • Energy charge – Range from 0 (all AMP) to 1 (all ATP) – Cell range from 0.8 to 0.95 • Higher EC favors biosynthesis and lower EC favors catabolism. • Alternative index: phoshorylation potential Phosphorylation potential = [ATP]/ [ADP]+[Pi] 70 Fig 15.19 Energy charge regulates metabolism •ATP-generating (catabolic) pathways are inhibited by a high energy charge, whereas ATP-utilizing (anabolic) pathways are stimulated by a high energy charge •High concentrations of ATP inhibit the relative rates of a typical ATPgenerating (catabolic) pathway and stimulate the typical ATP-utilizing (anabolic) pathway 71 • Controlling accessibility of substrates – Metabolic regulation and flexibility are enhanced by compartmentalization • Compartmentalization segregates opposed reaction – Fatty acid oxidation takes place in mitochondria – Fatty acid synthesis takes place in cytoplasm – The flux of the substrates • Glucose breakdown can take place in many cells only if insulin is present to promote glucose into the cell • The transfer of substrates from one compartment of a cell to another (from cytoplasm to mitochondria) 72