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Carbohydrate Metabolism II
Biochemistry/Lab (CHEM 3650)
Nova Southeastern University
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Carbohydrate Metabolism II: Aerobic Respiration Lecture Notes (Can be used for MCAT review too)
Acetyl-CoA
Citric Acid Cycle/Krebs Cycle/tricarboxylic acid (TCA) cycle: occurs in mitochondria and functions to oxidize Acetyl-CoA to CO 2 and H 2 O o Cycle produces high-energy molecules: NADH & FADH 2 Acetyl-CoA can be obtained from the metabolism of carbohydrates, fatty acids and amino acids
Methods of Forming Acetyl-CoA
Pyruvate dehydrogenase complex: multienzyme compound that catalyzes the reactions which involved pyruvate entering the mitochondrion and subsequently be oxidized and decarboxylated o Three carbon pyruvate is cleaved into a two-carbon acetyl group and a carbon dioxide Irreversible reaction (glucose cannot be formed directly from Acetyl,CoA o In mammals, complex is made up of five enzymes: Pyruvate dehydrogenase (PDH) Dihydrolipoyl transacetylase Dihydrolipoyl dehydrogenase Pyruvate dehydrogenase kinase Pyruvate dehydrogenase phosphatase
Overall reaction is exergonic: negative delta G, and is inhibited by accumulation of Acetyl-CoA and NADH.
Coenzyme A (CoA): written as CoA-SH to show that it is a thiol (with an –SH group) o Acetyl-CoA forms from a covalent attachment between the acetyl group and the –SH group. Results in the formation of thioester o Thioesters are high-energy compounds which are necessary to drive other reactions forward. Pyruvate Dehydrogenase (PDH): pyruvate is oxidized to yield CO 2 , and the remaining two-carbon molecules bind covalently to thiamine pyrophosphate (Vitamin B 1 or TPP) o TPP is a coenzyme that is non-covalently bonded to TPP o Mg2+ is also required in this reaction
Work together to convert pyruvate to Acetyl-CoA
Regulate the actions of PDH
Dihydrolipoyl Transacetylase: two-carbon molecule that is bound to TPP is oxidized and transferred to lipoic acid o Lipoic acid is a coenzyme that is covalently bonded to the enzyme Disulfide group acts as an oxidizing agent, which creates the acetyl group o Acetyl group is bonded to lipoic acid by a thioester linkage o Dihydrolipoyl transacetylase then catalyzed the reaction involving CoA-SH & the newly formed thioester link Causes the transfer of an acetyl group, so that it forms Acetyl-CoA o Lipoic acid is left in reduced from Dihydrolipoyl dehydrogenase: Uses the coenzyme Flavin adenine dinucleotide (FAD) to reoxidize the lipoic acid. o Lipoic acid oxidation results in FAD being reduced to FADH 2 FADH 2 is reoxidized to FAD by reducing NAD+ to NADH
Glycolysis is the main contributor to the production of Acetyl-CoA, however, other pathways are also present. o The end goal is always the same: produce Acetyl-CoA so that it can be fed into the citric acid cycle Fatty Acid Oxidation (-Oxidation): Activation causes a thioester bond to form between carboxyl group of fatty acids and CoA-SH o Activated fatty acyl-CoA cannot cross the inner mitochondrial membrane This is circumvented be having the fatty acyl group transferred to carnitine via a transesterification reaction o Once acyl-carnitine crosses the membrane, the fatty acyl group can be transferred to a mitochondrial CoA-SH Carnitine’s main function is to carry acyl group from cytosolic CoA-SH to a mitochondrial CoA-SH o Beta-oxidation can occur once Acetyl-CoA is formed in the mitochondrial matrix Removes two-carbon fragments from the carboxyl end. Amino Acid catabolism: Amino acid loses its amino group via transamination o Carbon skeletons can then form ketone bodies o Only certain amino acids can do this, these are termed: ketogenic
Step 3 - -Ketoglutarate and CO 2 Formation Isocitrate is oxidized to oxalosuccinate by isocitrate dehydrogenase Oxalosuccinate is decarboxylated to produce -Ketoglutarate and CO 2 Isocitrate dehydrogenase is the rate-limiting enzyme of the citric acid cycle One carbon is lost in this and the first NADH is produced from the intermediates of the cycle.
Step 4 – Succinyl-CoA and CO 2 Formation Reactions are carried out by the -Ketoglutarate dehydrogenase complex o Similar mechanism, cofactors, and coenzymes to PDH complex -Ketoglutarate and CoA come together to produce a molecule of Carbon dioxide o Carbon dioxide is the last carbon lost from the cycle o NAD+ is reduced to NADH in this step
Step 5 – Succinate Formation Thioester bond on succinyl-CoA is hydrolyzed to form succinate and CoA-SH o Coupled to the phosphorylation of GDP to GTP o Reaction catalyzed by succinyl-CoA synthetase Synthetases create new covalent bonds with energy input Hydrolysis of thioester bond releases a large amount of energy, which powers the phosphorylation of GDP. Nucleosidedipohosphate kinase catalyzes a phosphate transfer from GTP to ADP
o This is the only ATP that is produced through the citric acid cycle, rest is produced in the electron transport chain Step 6 – Fumarate Formation Only step that does not take place in mitochondrial matrix, occurs on the inner membrane Succinate undergoes oxidation to yield fumarate o Reaction catalyzed by succinate dehydrogenase Succinate dehydrogenase is a flavoprotein since it is covalently bonded to FAD (electron acceptor) o Enzyme is an integral protein on the inner mitochondrial membrane FAD is reduced to FADH 2 during this reaction o Each molecule of FADH 2 pass the electrons that it carries to the electron transport chain. (Indirectly gives rise to the production of 1 ATP) FAD is electron acceptor since Succinate is not a powerful enough reducing agent to reduce NAD+ Step 7 – Malate Formation Hydrolysis of alkene bond in fumarate, which produces malate o Catalyzed by fumarase Only L-malate forms from this reaction, even though it is possible for D-malate to form Step 8 – Oxaloacetate Formed Anew Malate is oxidized to oxaloacetate o Catalyzed by malate dehydrogenase Third molecule of NAD+ is reduced to NADH during this step Oxaloacetate can then be used in the cycle again, and the maximum amount of high energy electron carriers have been produced
Net Result and ATP Yield
- Glycolysis yields two ATP and two NADH molecules (net 9 molecules)
- For a single glucose molecule, the net ATP yield for glycolysis and oxidative phosphorylation is 30-32 ATP
Regulation
Pyruvate Dehydrogenase Complex Regulation Citric acid cycle can be regulated upstream from its actual starting point. This happens when PDH is phosphorylated
Aerobic is the most efficient way of getting energy since it is conducted in the mitochondria. Anaerobic (such as glycolysis and fermentation) is conducted in the cytosol Mitochondria’s is specifically designed to harvest energy: o Citric acid cycle takes place in the mitochondrial matrix o Assemblies needed for oxidative phosphorylation is housed in the inner membrane o Inner membrane is assembled into folds called cristae (maximize surface area) Essential for generating ATP through the proton-motive force Final step in aerobic respiration is two steps: o Electron transport along the inner mitochondrial membrane o Generation of ATP via ADP phosphorylation Overall process of aerobic respiration: o Electron rich molecules, NADH & FADH 2 , transfer electrons to carrier proteins in the inner mitochondrial membrane o Electrons are given to oxygen in the form of hydride ions (H-) and H 2 O is formed o Simultaneously, energy is released from transporting electrons facilitates proton transport at three different locations. Protons moved from mitochondrial matrix into the intermembrane space of the mitochondria (creates a greater concentration of hydrogen ions, which is used to drive ATP production)
Electron Flow and Complexes
Formation of ATP requires energy (endergonic). This energy is provided by the exergonic electron transport pathway. I. – Two reactions are coupled Molecule with higher potential will be reduced, and other molecule will be oxidized. This reduction potential is what drives the transfer of electrons o NADH is a good electron donor and oxygen is a good electron acceptor
Complex I (NADH-CoQ oxidoreducatase) Involves the transfer of electrons from NADH to coenzyme Q Complex has over twenty subunits, but two important ones are: o Protein that has an iron-sulfur cluster o Flavoprotein that oxidize NADH Has a coenzyme (Flavin mononucleotide) that is covalently bonded to it FMN is similar in structure to FAD (Flavin adenine dinucleotide)
First step involves NADH transferring its electrons over to FMN o NADH oxidized to NAD+ and FMN is reduced to FMNH 2 Flavoprotein then becomes reoxidized by reducing the iron-sulfur subunit Reduced iron-sulfur subunit donates its electrons that it received from FMNH 2 to coenzyme Q o CoQ becomes CoQH 2 Proton pumping occurs at this site: four protons are moved into the intermembrane space
Complex II (Succinate-CoQ oxidoreductase) Transfers electrons to coenzyme Q (ubiquinone) o Receives electrons from succinate instead of NADH (like in complex I) Succinate is oxidized by FAD (FAD is converted FADH 2 ) o Succinate dehydrogenase is also responsible for this (like in citric acid) FADH 2 then gets reoxidized when it reduces an iron-sulfur protein Iron-sulfur protein is then reoxidized by reducing CoQ No hydrogen pumping occurs here (Does not contribute to proton gradient)
Complex III (CoQH 2 -cytochrome C oxidoreductase) Facilitates the transfer of electrons from CoQ to cytochrome c Two separate complexes in drawing are meant to illustrate the two subsequent reaction that take place. Actually occur in the same complex Involves the reduction and oxidation of cytochromes o Proteins with heme groups where iron Is reduced to Fe2+ and reoxidized to Fe3+
Only one electron is transferred per reaction, so two cytochromes are needed per CoA Main contribution of complex 3 is through the Q Cycle: o Two electrons are shuttled from a molecule of ubiquinol (COQH 2 ), near the intermembrane space, to a molecule of ubiquinone (CoQ) near the mitochondrial matrix. o Two different electrons are attached to heme moieties, which reduces two molecules of cytochrome c Assisted by sulfur/iron carrier o Through the Q cycle, four protons are displaced into the intermembrane space Q cycle increases the gradient of the proton-motive force across the inner membrane Complex IV (Cytochrome c oxidase) Facilitates the culminating step of the electron transport chain o The transfer of electrons from cytochrome c to oxygen Contains subunits of cytochrome a, cytochrome a 3 , and Cu2+ ions o Two cytochrome subunits make cytochrome oxidase Cytochrome oxidase is oxidized as oxygen is reduced to form water Two protons are moved across the membrane
Malate is able to cross the membrane into the matrix and then reverse the above reaction by the enzyme malate dehydrogenase o NADH formed from this reverse reaction can pass along its electrons to the ETC through complex 1 o Generates 2 ATP Recycling of malate: o Malate oxidizes to oxaloacetate by malate dehydrogenase o Oxaloacetate is transaminated to form aspartate (aspartate transaminase) o Aspartate crosses into cytosol and is then reconverted oxaloacetate
Oxidative Phosphorylation
ATP synthase is a protein complex that spans the entire inner mitochondrial membrane and protrudes into the matrix.
Chemiosmotic Coupling
F 0 Portion: portion of ATP synthase that spans the membrane o Proton-motive force interacts with this portion o Functions as an ion channel: protons travel through F 0 , along their gradient, and back into the matrix. Chemiosmotic Coupling: a process which allows the chemical energy of a gradient to be harnessed. Describes a direct relationship between the proton gradient and ATP synthesis. o I. - The ETC generates a high concentration of protons in the intermembrane space, the protons then flow into the matrix through the F 0 ion channels, and this release of energy is harnessed for the phosphorylation of ADP to ATP F 1 Portion: utilizes the energy released from the gradient to phosphorylate ADP to ATP Conformation Coupling: an alternative pathway that says that the mechanism between proton gradient and ATP synthesis is indirect o States that ATP is released by the synthase as a result of conformation changes that are caused by the gradient o F 1 portion is like a turbine: spins to facilitate the harnessing of energy o Less scientifically accepted
-220 kJ/mol of energy from the exergonic proton-motive force dissipation. Energy is used to drive phosphorylation reaction
Regulation
O 2 and ADP are the key regulators of oxidative phosphorylation o If O 2 is limited, then the rate of oxidative phosphorylation decreases and the concentration of NADH and FADH 2 increases o Accumulation of NADH inhibits the citric acid cycle Respiratory Regulation: coordinated regulation of the different pathways that are involved If there is adequate oxygen available, then the rate of oxidative phosphorylation is dependent on the availability of ADP o ADP allosterically activates isocitrate dehydrogenase This increases the rate of the citric acid cycle and the production of electron carrying compounds: NADH & FADH 2 Enzyme’s cause an increase the rate of electron transport and ATP synthesis.
Carbohydrate Metabolism II
Course: Biochemistry/Lab (CHEM 3650)
University: Nova Southeastern University
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