MSU BMB 461 - Chapter 16 – The Citric Acid Cycle

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Chapter 16 – The Citric Acid Cycle- Respiration – the aerobic phase of catabolismo Refers to a multicellular organism’s uptake of O2 and release of CO2 o Biochemists and cell biologists use the term to refer to the molecular processes by which cells consume O2 and produce CO2 – cellular respiration- Cellular respiration occurs in three major stageso Organic fuel molecules are oxidized to yield two-carbon fragments in the form of the acetyl group of acetyl-coenzyme Ao The acetyl groups are fed into the citric acid cycle, which enzymatically oxidizes them to CO2 The energy released is conserved in NADH and FADH2o These reduced coenzymes are themselves oxidized, giving up protons and electrons Electrons are transferred to O2 via a chain of electron-carrying molecules (the respiratory chain) The large amount of energy released is conserved as ATP by oxidative phosphorylation- Respiration is believed to have evolved much later than glycolysis- Citric acid cycle = tricarboxylic acid (TCA) cycle = Krebs cycle16.1 Production of Acetyl-CoA (Activated Acetate)- In aerobic organisms, glucose and other sugars, fatty acids, and most amino acids are oxidized to CO2 and H2O via the citric acid cycle and the respiratory chain- Before entering the cycle, the carbon skeletons are degraded to the acetyl group of acetyl-CoA- Pyruvate dehydrogenase (PDH) complex – a cluster of enzymes that oxidizes pyruvate to acetyl-CoA and CO2o Located in the mitochondria of eukaryotic cells and in the cytosol of bacteriaPyruvate Is Oxidized to Acetyl-CoA and CO2- Oxidative decarboxylation – the overall reaction catalyzed by the pyruvate dehydrogenase complexo An irreversible oxidation process in which the carboxyl group is removed from pyruvate as a molecule of CO2 and the two remaining carbons become the acetyl group of acetyl-CoAThe Pyruvate Dehydrogenase Complex Requires Five Coenzymes- Requires three enzymes and five coenzymes/prosthetic groupso Thiamine pyrophosphate (TPP)o Flavin adenine dinucleotide (FAD)o Coenzyme A (CoA)o Nicotinamide adenine dinucleotide (NAD)o Lipoate- Four different vitamins are vital components of this system:o Thiamine (in TPP)o Rivoflavin (in FAD)o Niacin (in NAD)o Pantothenate (in CoA)- NAD and FAD are electron carriers- TPP is the coenzyme of pyruvate decarboxylase- Coenzyme A has a reactive thiol group that is critical to its role as an acyl carrier- Thioesters – formed by acyl groups covalently linking to the thiol groupo Have high standard free energies and therefore have high acyl group transfer potential- Lipoate – a cofactor that has two thiol groups that can undergo reversible oxidation to a disulfide bondo Can serve as both an electron carrier and as an acyl carrier.The Pyruvate Dehydrogenase Complex Consists of Three Distinct Enzymes- The PDH complex contains three enzymes:o Pyruvate dehydrogenase (E1)o Dihydrolipoyl transacetylase (E2)o Dihydrolipoyl dehydrogenase (E3)- E2 is the point of connection for the prosthetic group lipoate, attached through an amide bond to the amino group of a Lys residue- E2 has three functionally distinct domains:o The amino-terminal lipoyl domaino The central E1- and E3-binding domaino The inner-core acyltransferase domain- The domains of E2 are separated by linkers, sequences of 20 to 30 amino acid residues, rich in Ala and Pro and interspersed with charged residues- The active site of E1 has bound TPP and that of E3 has bound FAD- Also part of the complex are two regulatory proteins, a protein kinase and a phosphoprotein phosphataseIn Substrate Channeling, Intermediates Never Leave the Enzyme Surface- The pyruvate dehydrogenase complex decarboxylates and dehydrogenates with five consecutive reactions:o C-1 of pyruvate is released as CO2 and C-2, which in pyruvate has the oxidation state of an aldehyde, is attached to TPP as a hydroxyethyl group Slowest -> rate-limiting The point at which the PDH complex exercises its substrate specificityo The hydroxyethyl group is oxidized to the level of a carboxylic acid (acetate)o The acetyl moiety produced in this oxidation-reduction reaction is first esterified to one of the lipoyl –SH groups, than transesterified to CoA to form acetyl-CoAo Steps 4 and 5 are electron transfers necessary to regenerate the oxidized form of the lipoyl group of E2 to prepare the enzyme complex for another round of oxidation- The electrons removed from the hydroxyethyl group derived from pyruvate pass through FAD to NAD+- Substrate channeling – when the intermediates of a multistep sequence never leave the complex. o Prevents theft of the activated acetyl group by other enzymes that use this group as substrate16.2 Reactions of the Citric Acid Cycle- To begin the first turn of the citric acid cycle, acetyl-CoA donates its acetyl group to the 4-carbon compound oxaloacetate to form the six-carbon citrate. o Citrate is then transformed into isocitrate, which is dehydrogenated with loss of CO2 to yield the five-carbon compound -ketoglutarateo This undergoes a loss of a second molecule of CO2 and yields the four-carbon compound succinateo Succinate is enzymatically converted in three steps into oxaloacetate, which is ready to go back through the cycle- In each turn of the cycle, one acetyl group enters as acetyl-CoA and two molecules of CO2 leaveo One molecule of oxaloacetate is used to form citrate and one molecule of oxaloacetate is regeneratedo Oxaloacetate is present in cells in very low concentrations- Four of the eight steps in the citric acid cycle are oxidations, in which the energy from the oxidations is conserved in the form of NADH and FADH2- Four- and five-carbon intermediates of the citric acid cycle serve as precursors for a wide variety of productso Cells replenish these intermediates through anaplerotic reactions- The citric acid cycle takes place entirely in the mitochondriao The mitochondrion is the site for most energy-yielding oxidative reactions and of the coupled synthesis of ATPo Mitochondria are the major site of ATP production in the dark In the daylight, chloroplasts produce most of the organism’s ATPo In bacteria, the enzymes of the citric acid cycle are in the cytosol, and the plasma membrane plays a role analogous to that of the inner mitochondrial membraneThe Citric Acid Cycle Has Eight Steps- Formation of Citrateo The first reaction is the condensation of acetyl-CoA with oxaloacetate to form citrate, catalyzed by citrate

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MSU BMB 461 - Chapter 16 – The Citric Acid Cycle

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