Krebs Cycle PDF

Cell Energy Production Cell & Energy The energy is fundamental for the cells to sustain all the activities to survive, growth, exert the physiological functions The energy is produced by processing nutrients ingested ...

Cell Energy Production Cell & Energy The energy is fundamental for the cells to sustain all the activities to survive, growth, exert the physiological functions The energy is produced by processing nutrients ingested  Cell & Energy 258 15 Metabolism: Basic Concepts and Design FATS POLYSACCHARIDES PROTEINS phosphorylation, which are the final common pathways in the oxi- The of dation generation of energy fuel molecules. AcetylfromCoAthe oxidation brings of foodprod- the breakdown Stage 1 ucts of proteins, takes place insugars, threeand fats into the citric acid cycle (also called stages: Fatty acids and Glucose and Amino acids the tricarboxylic acid (TCA) cycle or Krebs cycle), where they are glycerol other sugars completely oxidized to CO2. 1.Stages In the rst3stage 2 and are the(top topicpanel), large of Sections molecules 7 through in consist 13 and Stage 2 of manyfood are broken metabolic downand pathways intohundreds smaller units. This In this of reactions. Acetyl CoA chapter, we willissee the basic itprinciples process digestion, rendersthatthe underlie not only the CoA biochemistry of Sections 7 through 13, but also macromolecules in our meals into biochemically the remainder of the text. Just as a simple alphabet can be used to generate an unlimited Citric more manageable fragments. Proteins are number of books, the basic principles of biochemistry discussed in acid 2 CO2 hydrolyzed this chapter can beto the 20 amino manipulated acids, to allow a cell and an organism to cycle respond to a wide range ofare polysaccharides physiological hydrolyzed circumstances. to simple sugars Stage 3 such as glucose, and fats are hydrolyzed to fatty 8 e– O2 acids. This stage is strictly a preparation stage; 15.1 Energy Is Required to Meet Three Oxidative no useful energy is captured at this point. phosphorylation Fundamental Needs H2O Living organisms require a continual input of free energy for three ATP major purposes: (1) the performance of mechanical work in muscle contraction and cellular movements, (2) the active transport of mol- Figure 15.1 Stages of catabolism. The ecules and ions, and (3) the synthesis of macromolecules and other biomolecules fi  Cell & Energy 258 15 Metabolism: Basic Concepts and Design FATS POLYSACCHARIDES PROTEINS phosphorylation, which are the final common The generation of energy from the oxidation of food pathways in the oxi- dation of fuel molecules. Acetyl CoA brings the breakdown prod- Stage 1 takes place in three stages: ucts of proteins, sugars, and fats into the citric acid cycle (also called Fatty acids and Glucose and Amino acids the tricarboxylic acid (TCA) cycle or Krebs cycle), where they are glycerol other sugars completely 2. In theoxidized secondtostage CO2. (middle panel), these Stages 2 and 3small numerous are themolecules topic of Sections 7 through to are degraded 13 and a consist Stage 2 of many metabolic pathways and hundreds of reactions. In this Acetyl CoA few simple units that play a central role in chapter, we will see the basic principles that underlie not only the metabolism. biochemistry In fact, of Sections most 13, 7 through of them—sugars, fatty of the but also the remainder CoA text. Just as a glycerol, acids, simple alphabet can be used and several aminoto generate an unlimited acids—are Citric number of books, into converted the basic acetylprinciples CoA, the of biochemistry activated two-discussed in acid 2 CO2 this chapter can be manipulated to allow a cell and an organism to cycle carbon unit that is the fuel for the nal stages of respond to a wide range of physiological circumstances. Stage 3 aerobic metabolism. Some adenosine 8 e– triphosphate (ATP) is generated in the second O2 Oxidative 15.1 stage, Energy butIsthe Required amount to Meetcompared is small Three with phosphorylation Fundamental that obtained in Needs the third stage. H2O Living organisms require a continual input of free energy for three ATP major purposes: (1) the performance of mechanical work in muscle contraction and cellular movements, (2) the active transport of mol- Figure 15.1 Stages of catabolism. The ecules and ions, and (3) the synthesis of macromolecules and other biomolecules fi  Cell & Energy 258 15 Metabolism: Basic Concepts and Design FATS POLYSACCHARIDES PROTEINS phosphorylation, which are the final common pathways in the oxi- The of dation generation of energy fuel molecules. Acetylfrom CoAthe oxidation brings of foodprod- the breakdown Stage 1 ucts of proteins, takes place insugars, threeand fats into the citric acid cycle (also called stages: Fatty acids and Glucose and Amino acids the tricarboxylic acid (TCA) cycle or Krebs cycle), where they are glycerol other sugars completely oxidized to CO2. 3.Stages In the third3 are 2 and stage the (bottom panel),7ATP topic of Sections is 13 and consist through Stage 2 of manyproduced metabolic from the complete pathways oxidation and hundreds of acetylIn this of reactions. Acetyl CoA chapter, we will CoA. Thesee thestage third basic consists principles of that theunderlie not only the citric acid CoA biochemistry of Sections 7 through 13, but also cycle and oxidative phosphorylation, which are the remainder of the text. Just as a simple alphabet can be used to generate an unlimited Citric the nal common pathways in the oxidation number of books, the basic principles of biochemistry discussed inof acid 2 CO2 fuel molecules. this chapter Acetyl CoA can be manipulated brings to allow theand an organism to a cell cycle respond to a wide range breakdown of physiological products of proteins,circumstances. sugars, and Stage 3 fats into the citric acid cycle (also called the 8 e– O2 tricarboxylic acid (TCA) cycle or Krebs cycle), 15.1 Energy Is Required to Meet Three Oxidative where they are completely oxidized to CO2. phosphorylation Fundamental Needs H2O Living organisms require a continual input of free energy for three ATP major purposes: (1) the performance of mechanical work in muscle contraction and cellular movements, (2) the active transport of mol- Figure 15.1 Stages of catabolism. The ecules and ions, and (3) the synthesis of macromolecules and other biomolecules fi Why do we need energy from food? 1. the performance of mechanical work in muscle contraction and cellular movements; 2. the active transport of molecules and ions; 3. the synthesis of macromolecules and other biomolecules from simple precursors.  Anaerobic Aerobic ditions in the cell. They are referred to as amphibolic pathways. An important general principle of metabolism is that, although biosynthetic Energy pathways and degradative pathways often have reactions in common, the regulated, H OH O irreversible reactions of each pathway are almost always distinct from each other. C O C CoA H3C C H3C S – O Acetyl CoA Lactate Metabolism of Complex Carbohydrates Metabolism of Cofactors and Vitamins Fuels are degraded and large molecules are constructed Figure 15.2 Glucose metabolism. Glucose is metabolized to pyruvate in 10 step by step in a series of linked reactions called metabolic linked reactions. Under anaerobic conditions, pyruvate is metabolized to lactate and, under Nucleotide Metabolism of Complex Lipids Metabolism pathways. aerobic conditions, to acetyl CoA. The glucose-derived carbon atoms of acetyl CoA are subsequently oxidized to CO2. An energy currency common to all life forms, ATP, links energy-releasing pathways with energy-requiring pathways. Carbohydrate Metabolism Metabolism of The oxidation of carbon fuels powers the formation of ATP. Other Amino Acids Lipid Metabolism Amino Acid Although there are many metabolic pathways, a limited Metabolism number of types of reactions and particular intermediates are common to many pathways. Figure 15.3 Metabolic pathways. Each Energy Metabolism node represents a particular biochemical, and Metabolism of Metabolic pathways are highly regulated to allow the the lines represent reactions linking the chemicals. [From the Kyoto Encyclopedia of Other Substances e cient use of fuels and to coordinate biosynthetic Genes and Genomes (www.genome.ad.jp/kegg).] processes. ffi ATP ATP contains: A sugar (ribose) A nitrogenous base (adenine) A triphosphate group The energy is stored in energy-carrying molecules The most important is ATP, a nucleoside triphosphate  Cell & Energy The ATP hydrolysis is a process thermodynamically favored It is a multi-step process Cell & Energy  ATP Synthesis TWO ways: Substrate-levels phosphorylation Oxidative phosphorylation Some reactions in glycolysis and Krebs cycle There is a ux of electrons that store their energy in a form used to synthesize ATP fl  The two Players FAD & NAD Flavin Adenine Dinucleotide Nicotinamide Adenine Dinucleotide (NAD)  ATP Synthesis ATP production with glycolysis (Substrate-levels phosphorylation) is less e cient but about 100x faster than Oxidative Phosphorylation ffi  ATP Synthesis: what is the di erence? ATP production with glycolysis (Substrate-levels phosphorylation) is less e cient but about 100x faster than Oxidative Phosphorylation ff ffi Cell & Energy  sons reveal that ATP is not the only compound with a high phosphoryl-transfer efficiently as a carrier of phosphoryl groups. potential. In fact, some compounds in biological systems have a higher phosphoryl- transfer potential than that of ATP. These compounds include phosphoenol- ATP as a “cellular energy currency” !70 (16.73) pyruvate (PEP), 1,3-bisphosphoglycerate (1,3-BPG), and creatine phosphate COO! (Figure 15.7). Thus, PEP can transfer its phosphoryl group to ADP to form ATP. Indeed, this transfer is one of the ways in which ATP is generated in the breakdown !60 C O P Phosphoenolpyruvate (14.34) (PEP) of sugars (Chapter 16). Of significance is that ATP has a phosphoryl-transfer "G’° of hydrolysis in kJ/mol (kcal/mol) CH2 CH 3 potential that is intermediate among the biologically important phosphorylated molecules (Table 15.1). This intermediate position enables Figure 15.7 The ATP has a central position ATP to function role of ATP O C asO the P cellular energy currencyN H is illustrated N in phosphoryl-transfer reactions. The !50 !OOC (11.95) P efficiently as a carrier of phosphoryl groups. by its relation toCHOH role of ATP as the cellular energy currency is other phosphorylated compounds. illustrated by its relation to other C H C 2 NH CH2 O P !70 (16.73) phosphorylated compounds. ATP has a phosphoryl-transfer potential that is ATP has(9.56) a phosphoryl-transfer !40 1,3-Bisphosphoglycerate potential Creatine thatphosphate is intermediate (1,3-BPG) COO! intermediate among the biologically important phosphorylated molecules. among the biologically Adenine important Rib P P P phosphorylated molecules. !60 C O P Phosphoenolpyruvate High-phosphoryl-transfer-potential High-phosphoryl-transfer-potential compounds derived from (14.34) (PEP) !30 ATP HIGH-ENERGY "G’° of hydrolysis in kJ/mol (kcal/mol) compounds derived from the metabolism of (7.17) CH2 CH 3 are used to power ATP COMPOUNDS O O P fuel molecules !50 C !OOC H synthesis.NIn turn, N the metabolism ATP donates a phosphoryl of fuel molecules are used to power LOW-ENERGY atp group P !20 COMPOUNDS (11.95) C to otherCbiomolecules to facilitate synthesis. (4.78) Glucose 6- P Glycerol 3- P CHOH their H metabolism. [Data from D. L. Nelson and 2 NH M. M. Cox, Lehninger Principles of CH2 O P !40 (9.56) 1,3-Bisphosphoglycerate (1,3-BPG) Creatine phosphate Biochemistry, 5th ed. (W. H. Freeman and In turn, ATP !10 (2.39) donates a phosphoryl group to other Company, 2009), Fig. 13-19.] Adenine Rib P P P biomolecules to facilitate their metabolism. Table 15.1 Standard free energies of hydrolysis (∆G°′) of some phosphorylated !30 ATP HIGH-ENERGY compounds (7.17) COMPOUNDS Compound kJ mol−1 kcal mol−1 LOW-ENERGY !20 COMPOUNDS Phosphoenolpyruvate (PEP) −61.9 −14.8 (4.78) Glucose 6- P Glycerol 3- P 1,3-Bisphosphoglycerate (1,3-BPG) −49.4 −11.8 Creatine phosphate −43.1 −10.3 !10 (2.39) ATP (to ADP) −30.5 −7.3 Table 15.1 Standard free energies of hydrolysis (∆G°′) of some phosphorylated Glucose 1-phosphate −20.9 −5.0 compounds Pyrophosphate (PPi) −19.3 −4.6 Compound kJ mol−1 kcal mol−1 Glucose 6-phosphate −13.8 −3.3 Phosphoenolpyruvate (PEP) −61.9 −14.8 Glycerol 3-phosphate −9.2 −2.2 1,3-Bisphosphoglycerate (1,3-BPG) −49.4 −11.8 Creatine phosphate −43.1 −10.3  phosphoryl groups for ATP regeneration for activities that require quick bursts catalyzed by creatine kinase: of energy or short, intense sprints (Figure 15.8). The fact that creatine phosphate can replenish ATP pools is the basis of creatine’s use as a dietary Creatine kinase Exercise Depends on Various Means of Generating ATP Creatine phosphate + ADP 3::::::::4 ATP + creatine supplement by athletes in sports requiring short bursts of intense activity. After the creatine phosphate pool is depleted, ATP must be generated through In resting muscle, typical concentrations of these metabolites are metabolism (Figure 15.9). [ATP] = 4 mM, [ADP] = 0.013 mM, [creatine phosphate] = 25 mM, and [creatine] = 13 mM. Because of its abundance and high phosphoryl-transfer potential relative to that of ATP (Table 15.1), creatine phosphate is a highly 15.3 ATP Is effective phosphoryl buffer. Indeed, creatine phosphate is the major source of At rest, muscle contains only enough ATP to sustain CLINICAL INSIGHT phosphoryl groups for ATP regeneration for activities that require quick bursts of energy or short, intense sprints (Figure 15.8). The fact that creatine phosphate can replenish ATP pools is the basis of creatine’s use as a dietary contractile activity for less than a second. Creatine supplement by athletes in sports requiring short bursts of intense activity. Exercise Depends on Various Means of Generating ATP After the creatine phosphate pool is depleted, ATP must be generated through metabolism (Figure 15.9). phosphate, a high-phosphoryl-transfer-potential molecule in At rest, muscle contains only enough ATP to sustain contractile activity for vertebrate muscle, serves as a reservoir of high-potential less than a second. Creatine phosphate, a high-phosphoryl-transfer-potential phosphoryl molecule groups in vertebrate that muscle, can as serves bea reservoir readily transferred to ADP. of high-potential phosphoryl groups that can be readily transferred to ADP. This reaction is catalyzed by creatine kinase: This reaction is catalyzed by creatine kinase: Figure 15.8 Sprint to the finish. Creatine phosphate is an energy source for intense Figure 15.8 Sprint to the finish. Creatine Creatine kinase phosphate is an energy source for intense Creatine phosphate + ADP ATP + creatine sprints. [David Stockman/AFP/Getty Images.] 3::::::::4 sprints. [David Stockman/AFP/Getty Images.] ATP Aerobic metabolism (Sections 8 and 9) ATP Aerobic metabolism In resting muscle, typical concentrations of these metabolites are Creatine phosphate [ATP] = 4 mM, [ADP] = 0.013 mM, [creatine phosphate] = 25 mM, and Figure 15.8 Sprint to the finish. Creatine Anaerobic (Sections 8 and 9) phosphate is an energy source for intense metabolism sprints. [David Stockman/AFP/Getty Images.] [creatine] = 13 mM. Because of its abundance and high phosphoryl-transfer Energy (Section 7) Figure 15.9 Sources of ATP during Creatine ATP phosphate Aerobic metabolism (Sections 8 and 9) potential relative to that of ATP (Table 15.1), creatine phosphate is a highly exercise. Exercise is initially powered by existing high-phosphoryl-transfer Anaerobic compounds (ATP and creatine phosphate). effective phosphoryl buffer. Indeed, creatine phosphate is the major source of metabolismSubsequently, the ATP must be regenerated Energy Creatine phosphate Seconds Minutes Hours Anaerobic metabolism phosphoryl groups (Section 7)by metabolic pathways. for ATP regeneration Figure 15.9 Sources of ATP during for activities that require quick bursts Energy (Section 7) Figureof15.9energy Sources of ATPorduring short, intense exercise. sprints Exercise (Figure is initially 15.8). The fact that creatine powered by exercise.Exercise is initially powered by phosphate can replenish existing high-phosphoryl-transfer compounds (ATP and creatine phosphate). ATP pools is the basis of creatine’s use as a dietary existing high-phosphoryl-transfer compounds (ATP and creatine phosphate). Seconds Minutes Hours supplement by athletes Subsequently, the ATP must be regenerated by metabolic pathways. in sports requiring short bursts of intense activity. Subsequently, the ATP must be regenerated Seconds Minutes Hours After the creatinebyphosphate pool is depleted, ATP must be generated through metabolic pathways. metabolism (Figure 15.9). Cell & Energy production The ATP reserve is limited (a few grams) The mean amount of ATP required per day is 83 kg (male of 70 kg) The recycle rate (ADP to ATP) is very high (300 times/mol ATP) Convergence of Energy Pathways The catabolism of carbohydrates, lipids and proteins converge into a common pathway: the Krebs Cycle This Cycle can occur in the presence of oxygen, because it can only work when also the next step, which is the oxidative phosphorylation can take place.  lated by ✓2 Identify the means by which the pyruvate dehydrogenase complex is regulated. multiple allo- From glucose to acetyl CoA ucose can be 4). However, Glucose animals and idative decar- of glucose to The oxidative decarboxylation of pyruvate to cycle with the acetyl CoA commits the carbon atoms of glucose ecause acetyl e 18.8). High Pyruvate to either of two principal fates: A inhibits the ADH inhibits Pyruvate (1) oxidation to CO2 by the citric acid cycle with dehydrogenase DH and acetyl complex the concomitant generation of energy n met or that d degradation Acetyl CoA (2) incorporation into lipid, because acetyl CoA is vate to acetyl st pyruvate is a key precursor for lipid synthesis s is covalent horylation of enase (PDH) CO2 Fatty acids sed by the ac- osphatase are Figure 18.8 From glucose to acetyl  Krebs Cycle and its two main functions The Krebs cycle can be used to produce ATP but its intermediates can be used to synthesise important biomolecules  C N N P P H3C S C O O Convergence of Energy Pathways O CH3 CH3 O Acetyl coenzyme A (Acetyl CoA) O 2 H3C C Acetyl unit The 18.1 Figure catabolism of A.carbohydrates, Coenzyme Coenzyme A is the activated ca fuel lipids for the and proteins citric acid converge cycle, is formed byinto a the pyruvate dehydro common pathway: the Krebs Cycle Four-carbon Six-carbon This Cycle can occur in the presence acceptor molecule presence of oxygen (aerobic conditions), of oxygen, because it can only work it is con acetyl coenzyme A (acetyl CoA; Figure when also the next step, which is the 18.1), tha ATP 2 CO2 cycle. The path that pyruvate takes oxidative phosphorylation can take depends on the oxygen availability. place. In most tissues, pyruvate is pro gen is readily available. For instance, in resting hu processed aerobically by first being converted into active muscle, for instance, the thigh muscles of a s High-transfer-potential electrons processed to lactate because the oxygen supply can Figure 18.2 An overview of the citric A schematic portrayal of the citric acid cycle acid cycle. The citric acid cycle oxidizes citric acid cycle accepts two-carbon acetyl units in two-carbon units, producing two molecules Cell & Energy production Overview on the sites, processes and yield of ATP in cells. Krebs cycle - The Krebs cycle (also named citric acid cycle, or tricarboxylic acid cycle) is a metabolic process that oxidate glucose derivatives to carbon dioxide, to generate ATP. - It represents the nal step for the catabolism of fuel molecules (carbohydrates, fatty acids and amino acids). - It is also important for the production of precursors of many other molecules such as amino acids, nucleotide bases, and porphyrin The step before the OXPHOS is the Krebs cycle, a cyclic series of reactions fi Krebs cycle: a cyclic pathway Unlike glycolysis, the citric acid cycle is a closed loop: the last part of the pathway regenerates the compound used in the rst step. The eight steps of the cycle are a series of redox, dehydration, hydration, and decarboxylation reactions that produce two carbon dioxide molecules, one GTP/ATP, and reduced forms of NADH and FADH2. This is considered an aerobic pathway because the NADH and FADH2 produced must transfer their electrons to the next pathway in the system, which will use oxygen. If this transfer does not occur, the oxidation steps of the citric acid cycle also do not occur. fi  Krebs cycle - It takes place in the matrix of the mitochondria under aerobic conditions - The pyruvate from glycolysis enters in mitochondria as Acetyl coenzyme A (Acetyl CoA), after an oxidative decarboxylation The Krebs Cycle  Krebs cycle - Through a series of oxidation-reduction reactions, high energy electrons are generate and used to produce ATP - Brie y, a C4 compound (oxaloacetate) condenses with an 1 acetyl to give a C6-tricarboxylix acid. The two following oxidative decarboxylation generates high-energy electrons. 2 - The electrons are used to reduce NAD+ and FAD to NADH and FADH2. - Their reoxidation releases electrons that generate a proton gradient across the inner mitochondrial m e m b r a n e , u s e d t o g e n e r a t e AT P ( o x i d a t i v e phosphorylation) fl Pyruvate and its central role in metabolism Pyruvate plays a central role, as it is needed for the synthesis of oxaloacetate Pyruvate Alanine Cysteine Glycine Serine Glucose Threonine Tryptophan Glycolysis Pyruvate Aerobic Anaerobic conditions conditions Mitochondria Cytosol Oxidative Lactic decarboxylation fermentation Fatty acid oxidation Acetyl-CoA Lactic Acid Krebs Cycle Pyruvate  Pyruvate fates T. Bender, J.-C. Martinou / Biochimica et Biophysica Acta 1863 (2016) 2436–2442 2437 Alternative fates of pyruvate at the pyruvate branch for about four decades. Here, we will not summarize the early attempts to identify and to characterize the MPC, because they are covered in point. The critical metabolite pyruvate is either excellent previous reviews to which the reader is referred [9,10]. In 2012, two seminal studies simultaneously reported identification of reduced in the cytosol by lactate dehydrogenase the MPC, although neither group had originally intended to study this (LDH), or imported into the mitochondrial matrix by transporter [3,4]. Rather, the original goal of both groups had been the characterization of two paralogous proteins of unknown function, the mitochondrial pyruvate carrier (MPC). known as BRP44L and BRP44 in mammals, that had attracted attention because they are highly conserved from yeast to humans, thus implying Inside mitochondria, two enzymes direct pyruvate a fundamental cellular function. Bricker et al. discovered defects in py- ruvate metabolism in a mutant of the Drosophila ortholog , while towards di erent downstream pathways. The Herzig et al. found perturbations in lipoic acid metabolism in yeast dele- tion mutants, pyruvate via Acetyl-CoA being the precursor for biosyn- pyruvate dehydrogenase complex (PDH) oxidizes thesis of this important cofactor. Both groups reached the same pyruvate to Acetyl-Co, which then enters the TCA conclusion that the two genes encode subunits of the MPC, and the pro- teins were renamed accordingly as MPC1 and MPC2. A third subunit ex- cycle, ultimately fueling the respiratory chain to ists in Saccharomyces cerevisiae (baker's yeast), which is called Mpc3, and whose sequence is almost identical to yeast Mpc2, suggesting that produce ATP. these proteins may be functionally related (see below and Fig. 3A). Subsequent experiments have demonstrated that MPC1 and MPC2 Pyruvate carboxylase (PC) catalyzes the in mammalian cells, as in yeast, are integral mitochondrial inner mem- brane proteins [3,4]. Two lines of evidence suggest that the MPC pro- conversion to oxaloacetate, which in addition to its teins are both necessary and sufficient for mitochondrial pyruvate uptake: (i) mitochondria isolated from mpc deletion mutants fail to im- anaplerotic role is also a precursor for port radioactively labeled pyruvate, and (ii) mouse MPC1 and MPC2, gluconeogenesis in hepatocytes. when co-expressed heterologously in Lactococcus lactis, allowed inhibitor-sensitive pyruvate uptake into these bacteria [3,4]. Further- more, mpc deletion mutants in yeast display a growth defect in minimal medium lacking branched-chain amino acids [3,4], most likely because Fig. 1. Alternative fates of pyruvate at the pyruvate branch point. The critical metabolite in fungi mitochondrial pyruvate is the precursor for the biosynthesis ff Mitochondrial Pyruvate Carrier Function in Health and Disease Biomolecules. 2020 Aug; 10(8): 1162. Pyruvate oxidative decarboxylation Pyruvate is carried to the mitochondria and oxidatively decarboxylated by the pyruvate dehydrogenase complex The reaction is irreversible Krebs Cycle: the overall reactions Pyruvate oxidative decarboxylation Pyruvate is carried to the mitochondria and oxidatively decarboxylated by the pyruvate dehydrogenase complex Pyruvate + CoA + NAD+ Acetyl CoA + CO2 + NADH + H+ The reaction is irreversible The complex is formed by three enzymes: The reaction needs ve coenzymes: 1. Pyruvate dehydrogenase component (E1) 1. Thaimine pyrophosphate (TPP) 2. Dihydrolipoyl transacetylase (E2) 2. Lipoic acid 3. Dihydrolipoyl dehydrogenase (E3) 3. FAD 4. CoA 5. NAD+ fi Regulation of pyruvate dehydrogenase PDH is regulated allosterically by the energy balance through i) its own products (negative feedback); ii) its own substrates (feed forward), iii) ATP and ADP Acetil CoA synthesis AcCoA is also generated through other sources: fatty acid, ketone bodies, some aminoacids Leu, Lys, Phe, Trp, Tyr Ile, Leu, Trp Citrate synthesis Citrate synthase ΔG°′ =−31,4 kJ/mol Aldol condensation (aldol reaction) between oxaloacetate (4C) and acetyl CoA (2C) to form citrate (6C), catalyzed by citrate synthase. The equilibrium reaction is toward the products because the thioester bond is energy rich. O x a l o a c e t a t e d e r i v e s f ro m t h e pyruvate carboxylation and it is also regenetated at the end of every Krebs cycle. Citrate synthesis The peculiar structure of the enzyme minimizes the side reaction (i.e. hydrolysis of acetil CoA). The binding of oxaloacetate causes a conformational change that create the acetyl CoA binding site. The new conformation puts the reagents in proximity, allowing the condensation. Citrate synthesis Oxaloacetate rst condenses with acetyl CoA to form citryl CoA, a molecule that is energy rich because it contains the thioester that originated in acetyl CoA. fi  Citrate synthesis 19.2 Stage One Oxidizes Two Carbon Atoms to Gather Energy-Rich Elect CoA S H2O CoA COO– oA S C H2C H2C O C O HO C COO– – H3C HO C COO CH2 –OOC CH2 –OOC Acetyl CoA Citryl CoA Citrate n is catalyzed by citrate synthase. Oxaloacetate first condenses DID The citrate syntheses is inhibited by NADH(H+) and by succinyl-CoA (two following YOUofKNOW? products the cycle) to form citryl CoA, a molecule that is energy rich because it con- Citrate isomerization Aconitase ΔG°′ = +6,3 kJ/mol The isomerization is a two step reaction: 1. Dehydratation, forming cis-aconitate 2. Hydratation The aim of the reaction is to move the hydroxyl group (C3) to facilitate the carboxylation The enzyme is an iron-sulfur protein. Citrate isomerization Water removal from citrate and subsequent addition to cis- aconitate are catalyzed by the iron-sulfur center: sensitive to oxidative stress. Isocitrate oxidative decarboxylation Isocitrate dehyrogenase ATP and NADH + H+ IDH could be in two forms: 1. Dissociated = inactive ADP and NAD+ 2. Associated = active Associated Dissociated  Isocitrate oxidative decarboxylation IDH is an allosteric enzyme regulated by NADH (negative), ADP (positive) and Ca++ (positive) Isocitrate, NAD+ and NADH bind to ac ve sites. ADP and Ca++ bind to two dis nct allosteric sites In absence of ADP, is coopera ve toward Isocitrate (sigmoid curve) In presence of ADP, the curve change to hyperbole Ac va on depends strongly on[ ADP]: small change of [ADP], results in signi cant change in ac vity ti ti ti ti ti fi ti  -ketoglutarate oxidative decarboxylation -ketoglutarate dehydrogenase -ketogluatarate + NAD+ + CoA Succinyl-CoA + CO2 + NADH + H+ The reaction is very similar to the pyruvate decarboxylation There is the formation of a thioester linkage with CoA with a high transfer potential 𝛂 𝛂 𝛂  -ketoglutarate oxidative decarboxylation Regulation -ketoglutarate dehydrogenase is regulated by its own products NADH and SuccinylCoA (negative feedback), and activated by Ca++ 𝛂 𝛂 A conserved mechanism for oxidative decarboxylation. The pathways shown employ the same five cofactors (thiamine pyrophosphate, coenzyme A, lipoate, FAD, and NAD+), closely similar multienzyme complexes, and the same enzymatic mechanism to carry out oxidative decarboxylations of pyruvate (by the pyruvate dehydrogenase complex), α- ketoglutarate (in the citric acid cycle), and the carbon skeletons of the three branched-chain amino acids, isoleucine (shown here), leucine, and valine. Succinate generation from succinyl-CoA Succinyl-CoA synthetase GDP GTP The cleavage of the thioester bond is coupled to the phosphorylation of a purine nucleoside, GDP or ADP. It is the only step that generates a compound with high phosphoryl-transfer potential. In liver the GTP production is higher, in skeletal and heart muscle, the ATP synthesis is predominant. GTP is used to power the synthesis of succinyl CoA, which is a precursor for heme synthesis. Guanosine triphosphate (GTP) conversion to ATP The nucleoside diphosphokinase catalyzes the transfer of the phosphoric group from GTP to ADP guanine adenosine GTP and ATP di er for the purine, GTP has guanine ff Succinate generation from succinyl-CoA GTP genera on Substrate level phosphoryla on Energy of thioester allows for incorpora on of inorganic phosphate Goes through a phospho-enzyme intermediate ti ti ti Three nal steps: oxalacetate is regenerated 19.3 Stage Two Regenerates Oxaloacetate and Harvests Energy-Rich Electrons transfers the phosphate to ADP to generate ATP is called substrate-level phos- by phorylation. oxidation of succinate Recall that glycolysis forms ATP with substrate-level phosphoryla- tion reactions (Chapter 16). In Section 9, we will examine how ion gradients can be used to power ATP formation. Oxaloacetate Is Regenerated by the Oxidation of Succinate Succinate is subsequently oxidized to regenerate oxaloacetate. COO– FAD FADH2 COO– NAD+ NADH + H+ COO– H2O O COO– H C H C H C HO C H H C H C H C H H C H – H OOC COO– COO– COO– Succinate Fumarate Malate Oxaloacetate The reactions constitute a metabolic motif that we will see again in fatty acid degradation (Chapter 27) and synthesis (Chapter 28). A methylene group (CH2) fi Succinate oxydation Succinate dehydrogenase The enzyme is embedded in the inner mitochondrial membrane (Complex II) Inhibited by oxaloacetate FAD is the hydrogen acceptor because it requires less energy to be reduced The enzyme is an iron-sulfur protein (as aconitase) Fumarate hydration Fumarase The reaction is reversible The addition of H+ and OH- is stereospeci c, so only the L isomer is formed fi Malate oxydation Malate dehydrogenase Another NADH + H+ molecule is produced The reaction is driven by the use of the products: oxaloacetate by citrate synthase and NADH by the electron-transport chain  Krebs cycle Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O 2 CO2 + 3 NADH + 3 H+ + FADH2 + GTP + CoA The cycle is highly e cient because all the enzymes are physically associated, allowing the direct passage of the molecules from one active site to the next through The NADH and FADH2 generated, through the electron-transport chain, will generate 2.5 ATP per NADH and 1.5 ATP per FADH2, respectively. Each cycle produces 10 ATP ffi  Krebs cycle COO– O CoA CH2 H2O + C H3C S-CoA –OOC C OH COO– Citrate CH2 Aconitase H C OH O COO– synthase COO– C –OOC C H Citrate CH2 CH2 Isotope-labeling studies revealed that the two NADH + H+ COO– COO– NAD+ Oxaloacetate carbon atoms that enter each cycle are not Isocitrate Malate Isocitrate the ones that leave. NAD+ dehydrogenase dehydrogenase NADH + CO2 –OOC The two carbon atoms that enter the cycle as COO– C O HO C H the acetyl group are retained in the initial two CH2 CH2 CH2 decarboxylation reactions and then remain COO– COO– incorporated in the four-carbon acids of the Malate !-Ketoglutarate NAD+ !-Ketoglutarate + CoA cycle. Note that succinate is a symmetric Fumarase dehydrogenase complex molecule. CoA S C O H2O NADH + CO2 COO– Consequently, the two carbon atoms that H C CH2 C CH2 enter the cycle can occupy any of the carbon –OOC H Succinate dehydrogenase COO– Succinyl CoA synthetase Fumarate COO– positions in the subsequent metabolism of CH2 Succinyl CoA FADH2 CH2 ADP + Pi the four-carbon acids. FAD COO– ATP + CoA Succinate Figure 19.6 The citric acid cycle. Notice that, because succinate is a symmetric molecule, the identity of the carbon atoms from the acetyl unit is lost. Krebs cycle  The Krebs cycle regulation Pyruvate The r − ATP, acetyl CoA, the c and NADH not t + ADP and pyruvate Entry into the citric acid cycle is largely controlled down through pyruvate dehydrogenase, the enzyme that Acetyl CoA acid produces acetyl CoA. However, there are two ATP additional steps in the cycle that are subject to Oxalo- trate acetate regulation. These are the two steps in which carbon Citrate F on th dioxide molecules are released, and also the steps Malate is op at which the rst two NADH molecules of the cycle Isocitrate quire are produced. cycle Fumarate − ATP and NADH of th + ADP signi of hi Succinate α-Ketoglutarate ATP, A Succinyl − ATP, succinyl whic CoA, and CoA NADH enzy be ex Figure 19.7 Control of the citric acid nase fi  The Krebs cycle regulation Pyruvate The r − ATP, acetyl CoA, the c and NADH not t + ADP and pyruvate down Acetyl CoA acid ATP Oxalo- trate acetate Citrate F on th Malate is op Isocitrate quire cycle Fumarate − ATP and NADH of th + ADP signi of hi Succinate α-Ketoglutarate ATP, A Succinyl − ATP, succinyl whic CoA, and CoA NADH enzy be ex Figure 19.7 Control of the citric acid nase Krebs cycle regulation High energy charge = The control points are represented by the allosteric enzymes 1 Isocitrate dehydrogenase ADP, NAD+, Mg2+, isocitrate