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Chapter 15 The Tricarboxylic Acid Cycle In Bacteria : This happens in cytosol, which is regulated by the presence of oxygen In Eukaryotic cells : cytosol and mitochondria All stages are performed in mitochondria. The components of respiratory metabolism : 4 events for glucose catabolism : 1) Glycolytic pathway : production of pyruvate from glucose (in cytosol) 2) TCA cycle : oxidation of pyruvate to generate reduced electrons (coenzymes) (mitochondrial matrix) 3) Electron-transport chain : reoxidation of coenzymes at the expense of molecular oxygen (mitochondrial inner membrane) 4) Oxidative phosphorylation : generation of ATP from ADP using proton-gradient generated in the process of electron transport. (mitochondrial inner membrane) Typical efficiency of glucose : about 30 ATPs per glucose Steps in the TCA Cycle The Oxidative Decarboxylation of Pyruvate Leads to Acetyl-CoA The first event in the TCA cycle : entry of pyruvate to mitochondria. : highly conserved during evolution Generation of Acetyl-CoA by pyruvate dehydrogenase complex Pyruvate Dehydrogenase Complex : Three enzymes form a complex : Each of them contains TPP, lipoic acid or CoA as a cofactor : Pyruvate decarboxylase : Dihydrolipoyl transacetylase : Dihydrolipoyl dehydrogenase : perform oxidation of lipoic acid In Mammals : its molecular weight is 9 106 : It contains 60 of the transacetylases and 20-30 of the other two enzymes. Production of Acetyl-CoA : a) from pyruvate in mitochondria b) from oxidation of fatty acid : Acyl-CoA is transported to the space between outer mitochondrial membrane and inner mitochondrial membrane(It can not pass through the mitochondrial membrane) and pass through the inner mitochodrial membrane via formation of acyl-carnitine. The acyl-carnitine becomes acyl-CoA inside mitochodria, which is utilized for acetyl-CoA by -oxidation. : Carnitine acyltransferase I, carrior protein and carnitine acyltransferase II Multienzyme complexes in TCA cycle : pyruvate dehydrogenase, -ketoglutarate dehydrogenase Unaerobically growing bacteria in the absence of -ketoglutarate dehydrogenase GTP-utilizing steps in glucose metabolism : phosphoenolpyruvate carboxykinase (glyconeogenesis), succinate thiokinase (succinate synthetase: TCA cycle) Succinate synthetase : use GDP exclusively in animal tissues, however, some animals have isozyme using ADP in an exclusively manner with the GDP-specific enzyme. Action of phosphoenolpyruvate carboxykinase : generate phosphoenolpyruvate from oxaloacetate using 1 GTP (It is interesting to point out that one GTP would be generated from conversion of succinyl-CoA to succinate. Why GTP not ATP which is generated in mitochondria ? : There is a specific ATP transporting system out of mitochondria, which will keep the concentration of ATP inside the mitochondria low. Therefore, it is necessary to use another type of nucleotide to drive turn of TCA cycle in mitochondria.) Another interesting point : Formations of oxaloacetate from phosphoenolpyruvate and succinate from succinyl-CoA result in decrease of GDP and increase of TCA cycle pool. Cell Mol Life Sci 2002 Feb;59(2):213-9 Evidence of undiscovered cell regulatory mechanisms: phosphoproteins and protein kinases in mitochondria. Thomson M. School of Biological Sciences, The University of Sydney, New South Wales, Australia. The finding that mitochondria contain substrates for protein kinases lead to the discovery that protein kinases are located in the mitochondria of certain tissues and species. These include pyruvate dyhydrogenase kinase, branched-chain alpha-ketoacid dehydrogenase kinase, protein kinase A, protein kinase Cdelta, stress-activated kinase and A-Raf as well as unidentified kinases. Recent evidence suggests that mitochondrial protein kinases may be involved in physiological processes such as apoptosis and steroidogenesis. Additionally, the novel finding of low-molecular-weight GTP-binding proteins in mitochondria suggests the possibility that these may interact with mitochondrial protein kinases to regulate the activity of mitochondrial effector proteins. The fact that there are components of cellular regulatory systems in mitochondria indicates the exciting possibility of undiscovered systems regulating mitochondrial physiology. PMID: 11915939 [PubMed - indexed for MEDLINE] The Amphibolic Nature of the TCA Cycle Amphibolic : The TCA cycle is catabolic and anabolic. The intermediates which are coupled with other metabolisms are : oxaloacetate, -ketoglutarate, succinyl-CoA and citrate. (amphibolic) : oxaloacetate : for aspartate -ketoglutarate : glutamate succinyl-CoA : heme biosynthesis in animals. citrate : acetyl-CoA in cytosol for fatty acid synthesis Anaplerotic reactions to TCA cycle : pyruvate carboxylase reaction to provide oxaloacetate. Anaplerotic : the reactions to replenish the intermediates in a biochemical cycle (TCA cycle in this case) Oxidation of Other Substrates by the TCA Cycle The entry of carbohydrates and fats to the TCA cycle : the final products of these catabolism is acetyl-CoA The entry of amino acids to the TCA cycle : -ketoglutamate, succinyl-CoA and oxaloacetate. Oxidation of these intermediates needs supply of acetyl-CoA (or pyruvate). Some amino acids become pyruvate or acetyl-CoA The TCA Cycle Activity Is Regulated at Metabolic Branchpoints Two major functions of TCA cycle : 1. furnishing reducing equivalents(NADH, FADH2) to the electron-transport chain 2. providing substrates for biosynthesis pathways. Factors : TCA Cycle need two entry components, acetyl-CoA and oxaloacetate. Supply of these molecules must be regulated. And, the fate of citrate is also important. 1. Energy Charge : decrease the TCA cycle rate 2. NADH/NAD+ : decrease the TCA cycle rate 3. Acetyl-CoA : increase the pool size of TCA cycle intermediates The Main Regulatory Points : Both are related the above three factors. a. b. Partition of Pyruvate to Acetyl-CoA and Oxaloacetate : acetyl-CoA는 TCA cycle의 속도를 가속시키기는 하지만 전체의 농도를 증가시키는 효과(pool size를 증가시키는 효과)는 없다. 이런 관점에서 보면 pyruvate에서 oxaloacetate의 합성은 다른 경로(acetyl-CoA의 합성) 와 역할이 다르다. Citrate Pool : citrate와 isocitrate는 유사하게 보이지만 실재의 역할은 매 우 다르다. citrate의 경우 fatty acid synthesis에 관여하고 있고 또한 mitochondria membrane을 통과할 수 있지만 isocitrate는 그렇지 않다. Regulation of Amino Acids catabolism in TCA Cycle Entering TCA cycle of amino acids via -ketoglutarate and succinyl-CoA has no apparent regulation site during their conversion to oxaloacetate. : Since oxaloacetate can not go further without acetyl-CoA, pyruvate dehydrogenase will be responsible for regulation of amino acids. Electron Transport, Proton Translocation, and Oxidative Phosphorylation Electron Transport : Oxidation of NADH and FADH2 to secret proton out of the inner mitochondrial membrane. (generate proton gradient) Oxidative Phosphorylation : ATP formation utilizing the membrane potential generated proton gradient. Oxidative phosphorylation of glucose 1. Electron transport 2. Proton translocation 3. ATP synthesis Electron Transport Is a Membrane-Localized Process Outer membrane : semi-permeable Inner membrane : impermeable Cristae Bacteria : plasma membrane이 이 역할을 담당하고 있음. A Bucket Bridge of Molecules Carries Electrons from the TCA Cycle to O2 Cytochromes : a, b, c Cytochrome a, a3 Cytochrome bL, and bH Cytochrome c : water soluble, c1 Cytochrome oxidase : page 349 equation 3 Electron carrier의 구조 : flavoproteins, iron-sulfur proteins and ubiquinone 1) Flavoproteins : one or two-electron transfer NADH dehydrogenase(FMN), succinate dehydrogenase(FAD), Mitochondrial inner membrane : glycerol-3-phosphate dehydrogenase(FAD) Mitochondrial matrix space : at least eight other flavoproteins 2) Iron-sulfur proteins : one-electron carrier NADH dehydrogenase, succinate dehydrogenase 3) Ubiquinone : on inner membrane, two- or one-electron carriers In case of single electron carrier, semiquinones can be formed : anionic or neutral semiquinone depending on pH and on the nature of binding site when the semiquinone is bound to protein. Freely migrate on the membrane Most of the Electron Carriers Exist in Large Complexes 4 complexes, cytochrome c and ubiquinone(UQ) Complex I : NADH dehydrogenase complex Complex II : succinate dehydrogenase complex is the only enzyme in TCA cycle, which is embedded in the inner membrane. Formed FADH2 is used for electron transport directly whereas formed NADH in TCA cycle is a substrate of NADH dehydrogenase complex(complex I). Complex III : cytochrome bc1 complex Complex IV : cytochrome oxidase, Molecular oxygen radicals can be made as a intermediate., which is a main cause of aging of mitochondria. Ubiquinone and cytochrome c : electron transfer molecule between complexes Ubiquinone and cytochrome c can diffuse on the membrane. Ubiquinone diffuses in the membrane and cytochrome c diffuses on the membrane surface. Complex I and II Mediate the Transfer of Electrons From NADH and FADH 2 to Ubiquinone Complex I : proton이 membrane 밖으로 이동. complex II : proton의 translocation이 없음. Complex III, The Cytochrome bc1 Complex, Transfers Electrons from QH2 to Cytochrome While Translocating Protons by a Redox Loop Components : cytochrome bL and bH, an iron-sulfur protein Q cycle cytochrome bL and bH : UQH2의 regeneration, a redox loop The iron-sulfur protein : reduction of cytochrome c, a redox loop Complex IV, the Cytochrome Oxidase Complex, Transfers Electrons from Cytochrome c to O2 While Pumping Protons across the Membrane Cytochrome a, a3, copper ions Four one electron transfers to consume one molecule oxygen Electrons from Cytosolic NADH Are Imported by Shuttle Systems Cytosolic NADH In Plant : another NADH dehydrogenase In animal : NADH shuttle systems 1) Glycerol-3-phosphate dehydrogenase의 catalytic site는 mitochondria의 cytosolic site에 있음. 2) malate formation form oxaloacetate, followed by translocation into mitochondria and re-oxidation to oxalacetate : this process can be coupled with glutamate/aspartate shuttle Complete Oxidation of Glucose Yields about 30 Molecules of ATP