The citric acid cycle Sitruunahappokierto Citronsyracykeln Ove Eriksson BLL/Biokemia ove.eriksson@helsinki.fi Metabolome: The complete set of small-molecule metabolites to be found in a cell or an organism. Metabolomics: The study of the metabolome(s). 1
How to study the metabolome? Water-soluble or lipid-soluble metabolites are extracted from the tissue of interest. How to study the metabolome? The resulting mixture of metabolites are analyzed by mass spectrometry. How to study the metabolome? The resulting mixture of metabolites are analyzed by mass spectrometry. 2
How to study the metabolome? Network analysis: Each dot represents one metabolite and the lines all known possible ways to convert one metabolite to another. How to study the metabolome? Network analysis: Each dot represents one metabolite and the lines all known possible ways to convert one metabolite to another. Metabolite flux included. How to study the metabolome? Identifcation of disease -associated loci in the Networks Early diagnosis Drug design Personalized diet 3
Citric acid cycle =Citrate cycle =Krebs cycle =Tricarboxylate cycle =TCA-cycle What does the TCA-cycle do? Oxidize products from carbohydrate-, fat-, and amino acid metabolism to CO2 and NADH. Offer chemical building blocks for the synthesis of carbohydrates, fat, and amino acids. The TCA-cycle takes place in mitochondria Mitokondrio (kreikkaa) mitos = loimi khondrion = jyvä Mitochondria are found in all human cells except in the erythrocyte. There are about 10 000 mitochondria in one hepatocyte. There are 1-2 mitochondria in one chondrocyte. 4
Principal structure of a mitochondrion in 3D Outer membrane Inner membrane Intermembrane space Intracristal space Mitochondria from various tissues seen by electron microscopy 1 M Liver: all main metabolic pathways Heart: betaoxidation and oxidative phosphorylation Adrenal gland: steroid synthesis The ultrastructural differences reflect specialization Mitochondria from various tissues seen by electron microscopy 1 M Liver mitochondria: all major metabolic pathways 5
Mitochondria from various tissues seen by electron microscopy 1 M Liver mitochondria: all major metabolic pathways Mitochondria from various tissues seen by electron microscopy 1 M Heart: beta-oxidation and oxidative phosphorylation Mitochondria from various tissues seen by electron microscopy 1 M Heart: beta-oxidation and oxidative phosphorylation 6
The mitochondrial network by light microscopy Mitochondrial DNA is found in so called nucleoids Brown et al, Mol Cell Biol 2011, 56 The human nuclear genome encodes for 20 687 proteins. Mitochondria contain about 1500 proteins. 7
The mitochondrial genome encodes for 12-60 proteins, depending on species. (For humans 13 proteins). The remaining proteins are encoded by the nuclear genome. Most elements in biochemical building blocks are reduced. Living organisms Levande organismer -CH2-CH2- NH3 SH Early atmosphere CO2 N2, NO3, NO2 S, SO4 What does a (primitiv) cell need? A carbon source (i.e. CO 2 ) Protons and electrons to reduce the carbon (H +, e - ). Chemical energy for dehydration reactions (ATP). 8
One solution: sunlight is used to extract electrons and protons from water Living Levande organisms organismer Waste product H + e - O 2 Earth and atmosphere CO2 H2O The first global environmental catastrophe: large quantities of oxygen to the atmosphere Change direction of the reactions and we get respiration. -CH2-CH2- -CH2-CH2- CO2 H + e - O2 Chemical energy H2O 9
Catabolism, anabolism, and the redox state of carbon Fat, amino acids, phospholipids Carbohydrates Metabolic intermediates Metabolic intermediates Dissolved in body fluids Carbohydrates Catabolism and anabolism Nutrients (foodstuffs) Lipids Proteins Catabolic pathways: -biodegradation -converge TCA-cycle CO2 NADH Energy currency =ATP Carbohydrates Lipids Proteins Building blocks of cells and tissues Anabolic pathways: -biosynthesis -diverge Catabolism, anabolism and the redox state of carbon Catabolic pathways, oxidation Anabolic pathways, reduction NAD NADP NADH NADPH O2 ->H2O pentose phosphate oxidation 10
Catabolism, anabolism and the redox state of carbon Catabolic pathways, oxidation NAD NADH The citric acid cycle is a link between catabolism and anabolism Anabolic pathways, reduction NADP NADPH O2 ->H2O pentose phosphate oxidation Carbohydrates Catabolism and anabolism Nutrients Lipids Proteins Glucagon Catecholamines Catabolic pathways TCA-cycle CO2 NADH Anabolic pathways Insulin Carbohydrates Lipids Proteins Building blocks of cells and tissues Mitochondrial compartments Outer membrane-few proteins, freely permeable to ions and metabolites < 5000 Da. Intermembrane compartment - small volume. Inner membrane - extremely rich in proteins, impermeable except to some metabolites. Matrix - extremely high protein concentration, gel-like. 11
Mitochondrial compartments Porin, protein import machinery. Creatine kinase, cell death proteins. Substrate transporters, respiratory chain, protein import machinery. Citrate cycle, amino acid katabolism, beta-oxidation, mitochondrial DNA, mitochondrial ribosomes. TCA-cycle 12
Definition: Anaplerotic reactions increase the concentration of intermediates in the citric acid cycle. Cataplerotic reactions consume citric acid cycle intermediates. Anaplerotic reactions and cataplerotic reactions must be in balance If molecules are removed from the cycle for biosynthesis, then an equal number of molecules must be supplied by catabolic reactions. The basic chemistry of metabolic reactions is simple 1. Redox reactions - electrons are transferred between a metabolite and one of either: NAD/NADH NADP/NADPH FAD/FADH Ferredoxin Lipoic acid 13
The basic chemistry of metabolic reactions is simple 2. Hydration or dehydration coupled to group transfer. The spontaneous direction is hydrolysis in an aqueous environment. Dehydration requires input of energy from ATP. The basic chemistry of metabolic reactions is simple 2. Hydration or dehydration coupled to group transfer. The basic chemistry of metabolic reactions is simple 3. Isomerizations The number of atoms and electrons in the molecule remains the same. Requires no input of energy. Yields no energy. 14
Pyruvate dehydrogenase complex, PDH Lipoic acid, an essential coenzyme of pyruvate dehydrogenase complex 15
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Input of acetyl-coa leads to net oxidation in the cycle A condensation coupled to hydrolysis of a thioester bond. Input of acetyl-coa leads to net oxidation in the cycle Citrate can be exported from mitochondria and used for synthesis of fatty acids. 17
The aconitase reaction Isomerization 18
Isocitrate dehydrogenase Oxidation of isocitrate coupled to reduction of either NAD or NADP. Ketoglutarate dehydrogenase Redox reaction coupled to dehydrating condensation of CoA-SH and ketoglutarate. 19
Succinyl-CoA synthetase Dehydrating condensation of GTP or ATP coupled to hydrolysis of succinyl-coa. 20
Succinate dehydrogenase Oxidation of succinate coupled to reduction of FAD. Fumarase reaction Hydration 21
Fumarase reaction Malate can be exported from mitochondria. -> Gluconeogenesis from pyruvate Malate dehydrogenase reaction Oxidation of malate coupled to reduction of NAD. 22
Malate dehydrogenase reaction Oxaloacetate can be decarboxylated to phosphoenolpyruvate and exported from mitochondria. -> gluconeogenesis from lactate 23
Products of one turn of the TCA-cycle What happens with the NADH produced in the TCA-cycle? Answer: NADH reduces oxygen to water in the mitochondrial respiratory chain an this is coupled to ATP production. = oxidative phosphorylation Can mutations in TCA-cycle enzymes cause diseases? 24
Examples 1. Isocitrate dehydrogenase 2. Succinyl-CoA synthase 3. Succinate dehydrogenase and fumarase 1. Isocitrate dehydrogenase There are two different forms of isocitrate dehydrogenase using either NAD or NADP The NAD-dependent form is thought to be heterotetramer of 2,, and subunits The NADP-dependent form is monomeric Nature Genetics, 2008, 10, 1230 25
2. Succinyl-CoA synthase both ATP and GTP generating forms. Am J Hum Gen, 2007, 383 3. Succinate dehydrogenase and fumarase Mutations have been found in: 1. Succinate dehydrogenase. Locus: chromosome 11q23. 2. Fumarase. Locus: chromsome 1q42. 26
Mutations of SDH and fumarase Homozygous: severe neurological dysfunction. Early death. Heterozygous: cancer Succinate dehydrogenase - leiomyomatosis and renal cell carcinoma. Fumarase - extra-adrenal pheochromocytomas, so called paragangliomas. Paraganglioma of the carotid body Angiography of arteria carotis Tumor mass Paraganglioma of the carotid body Surgical removal of tumor Tumor mass Arteria carotis 27
The mechanistic link between the biochemical changes and the tumor remains unclear The main tasks of mitochondria: Maintain the citric acid cycle and related metabolic pathways. NADH oxidation,o2 reduction and oxidative phosphorylation. Assemble iron-sulphur centers. Steroid synthesis. Participate in the signal transduction of programmed cell death, apoptosis. What you should understand after this lecture: The position of the TCA cycle between catabolic and anabolic pathways. How substrates are fed into the TCA cycle for oxidation. How the TCA cycle is replenished with intermediates. How intermediates in the TCA cycle are used for biosynthesis. 28
Fatty acid synthesis THE END Glycolysis Krebs cycle Amino acid katabolism Beta oxidation 29