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Cellular Respiration Mitochondria : Outer membrane permeable to small molecules and ions Inner membrane highly impermeable; contains the respi...

Cellular Respiration Mitochondria : Outer membrane permeable to small molecules and ions Inner membrane highly impermeable; contains the respiratory chain for transfer of electrons to O2 and ATP synthase Matrix contains Krebs cycle enzymes, lipids and aminoacid oxidation enzymes Cellular Respiration Stage 1 oxidation of fuels to acetyl-CoA  Cellular Respiration Stage 2: acetyl groups oxidation in the citric acid cycle (tricarboxylic acid (TCA) cycle, Krebs cycle) Generates: 2 CO2 3 NADH 1 FADH2, 1 GTP  1 ATP Cellular Respiration Stage 3: electron transfer chain and oxidative phosphorylation NADH 2.5 ATP FADH2 1.5 ATP  Descarboxilação oxidante do piruvato pyruvate dehydrogenase (PDH) complex: cluster of 3 enzymes and 5 cofactors mitochondrial matrix 5 reacções intermediates remain bound to the enzyme subunits Pyruvate + CoA-SH + NAD+ → acetyl-CoA + CO2 + NADH + H+ Catalytic cofactors (used by PDH and regenerated): TPP, lipoamida and FAD Stoichiometric cofactors (used by PDH but not regenerated): CoA and NAD+  CoA-SH 1ª reacção: descarboxilação do piruvato 2ª reacção: oxidação do hidroxietil-TPP  3ª reacção: transferência do acetil 4ª e 5ª reacções: oxidação da dihidrolipoamida e do FADH2 The conversion of pyruvate into acetyl CoA by pyruvate dehydrogenase complex is the link between glycolysis and cellular respiration. Citrate synthase (ΔGº’ = -7.5 kcal/mol) Acetyl CoA is the fuel for the citric acid cycle. → All fuel molecules are ultimately metabolized to acetyl CoA or components of the citric acid cycle. History of citric acid cycle discovery Albert von Szent-Györgyi (1893-1986) Hans Adolf Krebs Otto Heinrich Warburg (1900-1981) (1883-1970) 1947: emigra para EUA 1933: emigra para RU Szent-Györgyi Verificou que o fumarato era completamente oxidado em CO2 e H2O; 1 micromole de fumarato resulta no consumo de 3 micromoles de oxigénio Sugeriu que os ácidos dicarboxílicos seriam catalisadores da oxidação de outras moléculas Krebs intermediários do ciclo do ácido citrico eram verdadeiros intermediários Krebs Oxigénio consumido (mmol) 88 (Açúcar + citrato) OBTIDO 66 (Açúcar + citrato) previsto 44 Açúcar 22 Oxidação completa do citrato 0 0 30 60 90 120 150 Tempo (minutos) original citric acid cycle (Nature rejects Krebs's paper, 1937)  Nobel Prize in Physiology or Medicine 1953 Lipmann assumed that Acetyl-phosphate, instead of acetyl-CoA, is the two-carbon “active” compound condensing with oxaloacetate CoA: 1945 Acetil-CoA: 1951 Hans Adolf Krebs Fritz Albert Lipmann (1900-1981) (1899-1986) NADH FADH2 1951 1945 1937 Acetil-CoA Hidratos de Carbono 1921 Ácidos Gordos / Proteínas Principais acontecimentos Descoberta do a-cetoglutarato (Martius e Knoop) Apresentado o Apresentado o ciclo do Ciclo da Ureia Proposto o Glioxilato (Krebs e Ciclo do ác. (Kornberg & Henseleit) Cítrico (Krebs) Krebs) 1931 1932 1937 1953 1957 Otto Warburg: Szent-Györgyi: Hans Krebs: Fritz Lipmann: Prémio Nobel Prémio Nobel Prémio Nobel Prémio Nobel pelo estudo pelos estudos na pela pela das “enzimas oxidação descoberta do descoberta da respiratórias” biológica e acção ciclo dos coenzima A da vitamina C ácidos tricarboxílicos Hans Adolf Krebs Um cientista com particular apetência pelos ciclos….  Mitochondria : Outer membrane permeable to small molecules and ions Inner membrane highly impermeable; contains the respiratory chain for transfer of electrons to O2 and ATP synthase Matrix contains Krebs cycle enzymes, lipids and aminoacid oxidation enzymes The Citric Acid Cycle Has Eight Steps citrate formed from acetyl-CoA and oxaloacetate is oxidized to yield: – 2 CO2 – 3 NADH – 1 FADH2 – 1 GTP or ATP Ciclo de Krebs Ciclo de Krebs  Formation of Citrate citrate synthase = catalyzes the condensation of acetyl- CoA with oxaloacetate to form citrate – involves the formation of a transient intermediate, citroyl-CoA – large, negative ∆G′° is needed because [oxaloacetate] is normally very low Formation of Isocitrate via Cis-Aconitate aconitase (aconitate hydratase) = catalyzes the reversible transformation of citrate to isocitrate through the intermediate cis- aconitate – addition of H2O to cis-aconitate is stereospecific – low [isocitrate] pulls the reaction forward  Oxidation of Isocitrate to α-Ketoglutarate and CO2 isocitrate dehydrogenase = catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate – Mn2+ interacts with the carbonyl group of the oxalosuccinate and stabilizes the transiently-formed enol – specific isozymes for NADP+ (cytosolic and mitochondrial) or NAD+ (mitochondrial) Oxidation of α-Ketoglutarate to Succinyl-CoA and CO2 α-ketoglutarate dehydrogenase complex = catalyzes the oxidative decarboxylation of α-ketoglutarate to succinyl- CoA and CO2 – energy of oxidation is conserved in the thioester bond of succinyl-CoA  Conversion of Succinyl-CoA to Succinate succinyl-CoA synthetase (succinic thiokinase) = catalyzes the breakage of the thioester bond of succinyl- CoA to form succinate – energy released drives the synthesis of a phosphoanhydride bond in either GTP or ATP (substrate level phosphorylation) Oxidation of Succinate to Fumarate succinate dehydrogenase = flavoprotein that catalyzes the reversible oxidation of succinate to fumarate – integral protein of the mitochondrial inner membrane in eukaryotes – contains three iron-sulfur clusters and covalently bound FAD  Hydration of Fumarate to Malate fumarase = catalyzes the reversible hydration of fumarate to L-malate – transition state is a carbanion Oxidation of Malate to Oxaloacetate L-malatedehydrogenase = catalyzes the oxidation of L-malateto oxaloacetate, coupled to the reduction of NAD+ – low [oxaloacetate] pulls the reaction forward – regenerates oxaloacetate for citrate synthesis  Aspectos do Ciclo de Krebs Citrato: pro-quiral Chemically identical carbons Alexander Ogston pointed out that “it is possible that an asymmetric enzyme which attacks a symmetrical compound can distinguish between its identical groups” Aspectos do Ciclo de Krebs Substrate channeling → metabolon Evidence is accumulating that the enzymes are physically associated with one another to facilitate substrate channeling between active sites. The word metabolon has been suggested as the name for such temporary multienzyme complexes. A metabolon is a temporary structural-functional complex formed between sequential enzymes of a metabolic pathway, held together by noncovalent interactions. Substrate channelling - The formation of metabolons allows passing the intermediary metabolic product from an enzyme directly as substrate into the active site of the consecutive enzyme of the metabolic pathway The Citric Acid Cycle Is Regulated at Three Exergonic Steps regulation occurs at strongly exergonic steps catalyzed by: – citrate synthase – isocitrate dehydrogenase complex – α-ketoglutarate dehydrogenase complex fluxes are affected by the concentrations of substrates and products: – end products ATP and NADH are inhibitory – NAD+ and ADP are stimulatory – long-chain fatty acids are inhibitory Regulação do ciclo de Krebs  [oxaloacetato] determina a velocidade global do ciclo In general, energy charge is the key: AMP/NAD+ activate ATP/NADH inhibit Microorganisms & Plants Why can plants and some microrganisms exist with acetate as sole energy source but humans cannot? Glyoxylate cycle Note: - Bypass the 2 decarboxylation steps of the CAC - 2 molecules of acetyl CoA enter per turn. → glucose Glyoxylate cycle enables plants, bacteria, fungi and Triacylglycerols protists to grow on acetate Após duas voltas do Ciclo: 4 Acetatos 1 Hexose + 2 CO2 Conversion of fatty acids to glucose Glyoxylate cycle enables plants, bacteria, fungi and protists to grow on acetate