BIOCH-9 2017 1 Aerobic metabolism 6.1) Citric Acid Cycle 6.2) Electron transport 6.3) Oxidative phosphorylation 6.4) Oxidative stress - Aerobic oxidation of glucose- greater amounth of energy then does fermentation - Oxygen highly reactive - Oxidative cell damage: enzymes, antioxidant molecules BIOCH-9 2017 2 The Citric Acid Cycle The citric acid cycle is the final common pathway for the oxidation of fuel molecules: amino acids, fatty acids, & carbohydrates. • Most fuel molecules enter the cycle as acetyl coenzyme A • This cycle is the central metabolic hub of the cell • It is the gateway to aerobic metabolism for any molecule that can be transformed into an acetyl group or dicarboxylic acid, • It is also an important source of precursors for building blocks • Also known as, Krebs Cycle, & Tricarboxylic Acid Cycle (TCA) Chapter 17: Outline 17.1 The citric acid cycle oxidizes two-carbon units 17.2 Entry to the cycle and metabolism through it are controlled 17.3 The cycle is a source of biosynthetic precursors BIOCH-9 2017 3 Overview of citric acid cycle 1. The function of the cycle is the harvesting of high-energy electrons from carbon fuels 2. The cycle itself neither generates ATP nor includes O2 as a reactant 3. Instead, it removes electrons from acetyl CoA & uses them to form NADH & FADH2 (high-energy electron carriers) 4. In oxidative phosphorylation, electrons from reoxidation of NADH & FADH2 flow through a series of membrane proteins (electron transport chain) to generate a proton gradient 5. These protons then flow back through ATP synthase to generate ATP from ADP & inorganic phosphate 6. O2 is the final electron acceptor at the end of the electron transport chain 7. The cytric acid cycle + oxidative phosphorylation provide > 95% of energy used in human aerobic cells BIOCH-9 2017 4 Fuel for the Citric Acid Cycle Thioester bond to acetate -mercapto-ethylamine Pantothenate Initiates cycle BIOCH-9 2017 5 Mitochondrion Double membrane, & cristae: invaginations of inner membrane BIOCH-9 2017 6 Mitochondrion Oxidative decarboxilation of pyruvate, & citric acid cycle take place in matrix, along with fatty acid oxidation Site of oxidative phosphorylation Permeable BIOCH-9 2017 7 Citric Acid Cycle: Overview Input: 2-carbon units Output: 2 CO2, 1 GTP, & 8 high-energy electrons BIOCH-9 2017 8 Cellular Respiration 8 high-energy electrons from carbon fuels Electrons reduce O2 to generate a proton gradient ATP synthesized from proton gradient BIOCH-9 2017 9 Glycolysis to citric acid cycle link Acetyl CoA link is the fuel for the citric acid cycle BIOCH-9 2017 10 Oxidative decarboxylation A large, highly integrated complex of three kinds of enzymes Pyruvate + CoA + NAD+  acetyl CoA + CO2 + NADH Groups travel from one active site to another, connected by tethers to the core of the structure -irreversible oxidation, carboxyl group is removed from pyruvate as CO2 + AcCoA BIOCH-9 2017 11 3 enzymes 5 Coenzymes: B1 vitamin Vitamines:thiamine, riboflavin (FAD), niacin (NAD), pantothenate (CoA) BIOCH-9 2017 12 CoA BIOCH-9 2017 13 Fuel for the Citric Acid Cycle Thioester bond to acetate -mercapto-ethylamine Pantothenate Initiates cycle BIOCH-9 2017 14 TPP Vitamin B1 BIOCH-9 2017 15 BIOCH-9 2017 16 BIOCH-9 2017 17 BIOCH-9 2017 18 BIOCH-9 2017 19 Citrate Cycle: step 1 (citrate formation) Enzyme: Citrate synthase Condensation reaction Hydrolysis reaction BIOCH-9 2017 20 Conformational changes in citrate synthase Homodimer with large (blue) & small (yellow) domains Open form Closed form BIOCH-9 2017 21 Citrate isomerized to Isocitate: step 2 Enzyme: aconitase Dehydration Hydration BIOCH-9 2017 22 Aconitase: citrate binding to iron-sulfur cluster 4Fe-4S iron-sulfur cluster BIOCH-9 2017 23 Isocitrate to -ketoglutarate: step 3 Enzyme: isocitrate dehydrogenase 1st NADH produced 1st CO2 removed Oxidation of IC to aKG and CO2, Mn2+ in active site BIOCH-9 2017 24 Succinyl CoA formation: step4 Enzyme: -ketoglutarate dehydrogenase 2nd NADH produced 2nd CO2 removed oxidation -SH BIOCH-9 2017 25 Succinate formation: step5 Enzyme: succinyl CoA synthetase GTP produced GTP + ADP  GDP + ATP (NPTase) BIOCH-9 2017 26 BIOCH-9 2017 27 Succinyl CoA synthetase Rossman fold binds ADP component of CoA ATP-grasp domain is a nucleotide-activating domain, shown binding ADP. His residue picks up phosphoryl group from near CoA, & swings over to transfer it to the nucleotide bound in the ATP-grasp domain BIOCH-9 2017 28 Oxaloacetate regenared by oxidation of succinate: Steps 6 - 8 Oxidation, hydration, and oxidation BIOCH-9 2017 29 Succinate to Fumarate: step 6 Enzyme: succinate dehydrogenase FADH2 produced BIOCH-9 2017 30 Fumarate to Malate: step 7 Enzyme: fumarase BIOCH-9 2017 31 Fumurate to L-Malate Hydroxyl group to one side only of fumarate double bond; hence, only L isomer of malate formed BIOCH-9 2017 32 Malate to Oxalate: step 8 Enzyme: malate dehydrogenase 3rd NADH produced BIOCH-9 2017 33 The citric acid cycle BIOCH-9 2017 34 Summary of 8 steps Proton gradient generates 2.5 ATP per NADH, & 1.5 per FADH2 9 ATP from 3 NADH + 1 FADH2. Also, 1 GTP Thus, 1 acetate unit generates equivalent of 10 ATP molecules. In contrast, 2 ATP per glucose molecule in anaerobic glycolysis BIOCH-9 2017 35 Pyruvate to Acetyl CoA, irreversible Key irreversible step in the metabolism of glucose BIOCH-9 2017 36 Regulation of pyruvate dehydrogenase Inhibited by products, NADH & Acetyl CoA Also regulated by covalent modification, the kinase & phosphatase also regulated BIOCH-9 2017 37 Control of citric acid cycle Allosteric regulation Regulated primarily by ATP & NADH concentrations, control points: Pyruvate dehydrogenase isocitrate dehydrogenase & - ketoglutarate dehydrogenase 2-oxo-glutarate dehydrogenase) Inhibition by product: citrate synthase- citrate- 2-oxo-glutarate dehydrogenasesuccinyl CoA BIOCH-9 2017 38 Biosynthetic roles of the citric acid cycle Chapter 16- e-book BIOCH-9 2017 Key Enzymes for regulation of CAC, inhibitors and activators Enzyme ATPa NADHa different Pyruvate dehydrogenase - - - acetyl-CoA (inh. prod.) Citrate syntethase - citrate (inhibition by product) Isocitrate dehydrogenase - - + ADP (allosteric activation) 2-OG-dehydrogenase - - sukcinyl-CoA (inh. prod.) 39 a allosteric inhibitor b feedback inhibitor (inhibition by reaction product) c allosteric activator NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 237. 39 Regulation of CAC BIOCH-9 2017 NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 238. 41 pyruvate citrat e Cis-aconitate isocitrate 2-oxoglutarate Succinyl-CoA Succinate oxalacetate oxalacetate fumarate 40 BIOCH-9 2017 41 BIOCH-9 2017 42 BIOCH-9 2017 pyruvate malate 43