Pyruvate Metabolism Lecture (2) PDF

Pyruvate metabolism 2023-2024 Dr. MOHANNED ALASADI Lecture (2) Formation of pyruvic acid (P.A.) in the body :  From oxidation of glucose (glycolysis).  From lactic acid by oxidation.  Deamination of Alanine.  Glucogenic amino acids-pyruvate forming.  Decarboxylation of oxa...

Pyruvate metabolism 2023-2024 Dr. MOHANNED ALASADI Lecture (2) Formation of pyruvic acid (P.A.) in the body :  From oxidation of glucose (glycolysis).  From lactic acid by oxidation.  Deamination of Alanine.  Glucogenic amino acids-pyruvate forming.  Decarboxylation of oxaloacetic acid (OAA) Fate of pyruvic acid (P.A.) 1 -Form acetyl CoA by oxidative decarboxylation (in presence of O2). 2 -Forms lactic acid by reduction (in absence of O2). 3 -Forms alanine by amination. 4 -Forms glucose (gluconeogenesis). 5 -Forms malic acid → to O.A.A (oxaloacetic acid). 6-Forms oxaloacetic acid (O.A.A) by CO2-fixation reaction. Pyruvate metabolism 3 Oxidative decarboxylation of pyruvate:  Before pyruvate can enter the TCA cycle, it must be transported into the mitochondria via a special pyruvate transporter that aids its passage across the inner mitochondrial membrane.  Within the mitochondria, pyruvate is oxidatively decarboxylated to acetyl-CoA, this reaction is catalyzed by sequentially multienzyme complex (pyruvate dehydrogenase complex).  The PDH complex is a protein aggregate of multiple copies of three enzymes, pyruvate carboxylase (E1, sometimes called pyruvate dehydrogenase), dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3).  The PDH complex contains five coenzymes that act as carriers or oxidants for the intermediates of the reactions E1 requires thiamine pyrophosphate (TPP), E2 requires lipoic acid and CoA, and E3 requires flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NAD+).  Regulation of pyruvate dehydrogenase (PDH) complex. The complex also contains two tightly bound regulatory enzymes, pyruvate dehydrogenase kinase (PDH kinase) and pyruvate dehydrogenase phosphatase (PDH phosphatase).  Mechanism of action of the pyruvate dehydrogenase complex 6  Pyruvate dehydrogenase (PDH). PDH requires thiamine pyrophosphate (TPP); this explains the serious afflictions in beriberi due to thiamine deficiency.  TPP deficiency in alcoholism causes pyruvate accumulation in tissues. Inherited PDH deficiency may lead to lactic acidosis.  Deficiencies of thiamine (B1) or niacin (B3) can cause serious central nervous system problems. This is because brain cells are unable to produce sufficient ATP (via the TCA cycle) if the PDH complex is inactive. Wernicke-Korsakoff, an encephalopathy-psychos is syndrome due to thiamine deficiency, may be seen with alcohol abuse. Leigh syndrome (subacute necrotizing encephalomyelopathy) is a rare, progressive, neurode -generative disorder caused by defects in mitochondrial ATP production, primarily as a result of mutations in genes that code for proteins of the PDH complex, the electron transport chain, or ATP synthase. Both nuclear and mitochondrial DNA can be affected.  Mechanism of arsenic poisoning  Arsenate can interfere with glycolysis at the glyceraldehyde 3-phosphate step, thereby decreasing ATP production.  “Arsenic poisoning” is, however, due primarily to inhibition of enzymes that require lipoic acid as a coenzyme, including E2 of the PDH complex, α-ketoglutarate dehydrogenase, and branched-chain α-keto acid dehydrogenase. Arsenite forms a stable complex with the thiol (–SH) groups of lipoic acid, making that compound unavailable to serve as a coenzyme. When it binds to lipoic acid in the PDH complex, pyruvate (and, consequently, lactate)  The citric acid cycle (TCA) 1-TCA cycle (tricarboxylic acid cycle), also known as the citric acid cycle or the Krebs cycle, is the major energy production pathways in the body. The cycle occur in the mitochondria. 2 -It is a cyclic process 3 -The cycle involves a sequence of compounds inter-related by oxidation reduction and other reaction which finally produces CO2 and H2O. 4 -It is the final common pathway of breakdown or catabolism of carbohydrates, fats and proteins. because glucose, fatty acid and most amino acid are metabolized to acetyl CoA or intermediate of the cycle 5-Acetyl CoA derived mainly from oxidation of either glucose or -oxidation of FA and partly from certain amino acids. 6 -By stepwise dehydrogenations and loss of two molecules of CO2, accompanied by internal re-arrangements, the citric acid is reconverted to OAA, which again starts the cycle by taking up another acetyl group from acetyl-CoA. 7- All the enzymes of the TCA cycle are in the mitochondrial matrix, which is in the inner mitochondrial membrane. 8- Electrons are transferred by the cycle to NAD+ and FAD. 9- As the electrons subsequently are passed to O2 by the electron transport chain, ATP is generated by the process of oxidative phosphorylation. 10- ATP is also generated from GTP, produced in one reaction of the cycle by substrate level phosphorylation. 11-The whole process is aerobic, requiring O2 as the final oxidant of the reducing equivalents. Absence of O2 (anoxia) or partial deficiency of O2 (hypoxia) causes total or partial inhibition of the cycle. 12-The H atoms removed in the successive dehydrogenations are accepted by corresponding coenzymes. Reduced coenzymes transfer the reducing equivalents to electron-transport system, where oxidative phosphorylation product ATP molecules. 10  There are three key enzymes in TCA cycle: 1-Citrate synthase 2- Isocitrate dehydrogenase(I.C.D) 3- α-ketoglutarate dehydrogenase  Biomedical importance of TCA cycle: - Final common pathway for carbohydrates, proteins and fats, through formation of 2 – carbon unit acetyl-CoA. - Acetyl-CoA is oxidized to CO2 and H2O giving out energy (Catabolic role). - Intermediates of TCA cycle play a major role in synthesis also like heme formation, formation of non essential amino acids, FA synthesis, cholesterol and steroid synthesis (anabolic role). TCA cycle is called Amphibolic in nature because TCA cycle has dual role catabolic and anabolic.  Role of Vitamins in TCA Cycle Five B vitamins are associated with TCA cycle essential for yielding energy.  Riboflavin B2: In the form of flavin adenine dinuleotide (FAD)— a cofactor for succinate dehydrogenase enzyme.  Niacin B3: In the form of nicotinamide adenine dinucleotide (NAD) the electron acceptor for isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and malate dehydrogenase.  Thiamine B1: As “thiamine diphosphate”—required as coenzyme for decarboxylation in the α-ketoglutarate dehydrogenase reaction.  Lipoic acid: It is required as coenzyme for α-ketoglutarate dehydrogenase reaction.  Pantothenic acid B5: As part of coenzyme A, the cofactor attached to “active” carboxylic acid residues such as acetyl CoA and succinyl-CoA.  Energy produced by the TCA cycle  The net reaction for the oxidation of one acetyl unit is : Acetyl - CoA + 3NAD+ + FAD + GDP + Pi → 2CO2 + 3NADH + 3H+ + FADH2 + GTP + CoA Energy – producing reaction Number of ATP produced  3NADH → 3NAD+ 3x3=9  FADH2 → FAD 2x1=2  GDP + Pi → GTP 1x1=1  Net gain : 12 ATP  Maximal ATP Production Overall, when 1 mole of glucose is oxidized to CO2 and H2O, approximately( 36 moles) of ATP are produced if the glycerol phosphate shuttle is used, or( 38 moles) if the malate aspartate shuttle is used. Note: Assuming each high energy bond to be equivalent to 7600 calories. Total energy captured in ATP per mol. of glucose oxidized = 7600× 38 = 288,800 calories. 18  Biological Oxidation  Oxidation is a reaction with oxygen directly or indirectly or to lose hydrogen and/or electrons. Biologically it is carried out by the enzymes.  Oxidation is defined as the loss of electrons and Reduction as the gain in electrons.  When a substance exists both in the reduced state and in the oxidized state, the pair is called a redox couple.  Biological Oxidation The transfer of electrons from the reduced coenzymes through the respiratory chain to oxygen is known as biological oxidation. Energy released during this process is trapped as ATP. This coupling of oxidation with phosphorylation is called oxidative phosphorylation. In the body, this oxidation is carried out by successive steps of dehydrogenations. Bioenergetics describes the transfer and utilization of energy in biologic systems. The change in free energy is represented in two ways, ΔG and ΔGo. The first, ΔG (without the superscript “o”), represents the change in free energy and, thus , the direction of a reaction at any specified concentration of products and reactants. ΔG, then, is a variable. This contrasts with the standard free energy change, ΔGo (with the superscript “o”), which is the energy change when reactants and products are at a concentration of 1 mol/l.  Reactions or processes that have a large positive ΔG, such as moving ions against a concentration gradient a cross a cell membrane , are made possible by coupling the endergonic movement of ions with a second, spontaneous process with a large negative ΔG such as the exergonic hydrolysis of adenosine triphosphate (ATP).  ATP consists of a molecule of adenosine (adenine + ribose) to which three phosphate groups are attached.  If one phosphate is removed, ADP is produced. If two phosphates are removed, adenosine monophosphate (AMP) results.  The standard free energy of hydrolysis of ATP, ΔGo, is approximately –7.3 kcal/mol for each of the two terminal phosphate groups.  Because of this large negative ΔGo, ATP is called a high- energy phosphate compound. Electron Transport Chain and Oxidative Phosphorylation -Electron Transport Chain: This is the final common pathway in aerobic cells by which electrons derived from various substrates are transferred to oxygen. - Electron transport chain(ETC) is a series of highly organized oxidation-reduction enzymes. The ETC is localized in the mitochondria. - Energy-rich molecules, such as glucose, are metabolized by a series of oxidation reactions ultimately yielding CO2 and water.  ATP is generated as a result of the energy produced when electrons from NADH and FADH2 are passed to molecular oxygen by a series of electron carriers, collectively known as the electron transport chain.  The components of the chain include FMN (Flavin mononucleotide), Fe-S centers, coenzyme Q, and a series of cytochromes (b, c1, c and aa3). The electron transport chain in the mitochondrial membrane has been separated on four (4) complexes, their components as follows: 1- Complex I : NADH – coenzyme Q Reductase. This system has two functions : -Electron transfer. -Acts as a proton pump.  NADH + H+ + FMN ⎯→ FMN.H2 + NAD+  2- Complex II : Succinate-Coenzyme Q Reductase. Flow of electrons from succinate to CoQ occurs via FADH2. Succinate + CoQ ⎯→ Fumarate + CoQ.H2 3- Complex III : Cyt.C Reductase. Function as :  Proton pump, and  Catalyzes transfer of electrons.  Fe+3 accepts electron and is oxidized to Fe+2  The energy change permits ATP formation.  Co.Q.H2 + 2 Cyt.C (Fe+3) ⎯→ Co.Q + 2 Cyt.C (Fe+2) + 2H+ 4- Complex IV : Cyt.C oxidase.  The system functions :  As proton pump.  Catalyzes transfer of electrons from Cyt.C to molecular O2 to form H2O via Cyt.a, Cu+2 ions and Cyt. a3. 4Cyt.C (Fe+2) + 4H+ + O2 ⎯→ 4 Cyt.C (Fe+3) + 2H2O The flow of electrons is as follows : Cyt. C ⎯→ Cyt. a ⎯→ Cu+2 ⎯→ Cyt. a3 ⎯→ O2  The energy change permits ATP formation between Cyt. a3 and molecular O2. The energy derived from the transfer of electrons through the electron transport chain is used to pump protons across the inner mitochondrial membrane from the matrix to the cytosolic side. -An electrochemical gradient is generated, consisting of a proton gradient and a membrane potential. -Protons moves back into the matrix through the ATP synthase complex, causing ATP to be produced from ADP and inorganic phosphate. -ATP is transported from the mitochondrial matrix to the cytosol in exchange for ADP (the ATP-ADP antiport system). (10)  ATP synthesis in mitochondria from ADP + Pi is catalyzed by ATP synthase (complex V) and driven by electron-transport process The free energy released by electron transport process must be conserved in a form that ATP synthase can use. This Chemiosmotic theory energy conservation is referred to as energy coupling. The free energy of electron transport is conserved by pumping H+ from the ATP synthesis is coupled to mitochondrial matrix to the electron-transport through the intermembrane space to create an formation of a transmembrane electrochemical H+ gradient across the inner mitochondrial membrane. The proton gradient during electron electrochemical potential of this gradient transport is harnessed to synthesize ATP. -The oxidation of NADH generates approximately( 3 ATP), while the oxidation of one FADH2 generates approximately ( 2 ATP). -Because energy generated by transfer of electrons through the electron transport chain to O2 is used in the production of ATP, the overall process is known as oxidative phosphorylation. Electron transport and ATP production occur simultaneously and are tightly coupled. -NADH and FADH2 are oxidized only if ADP is available for conversion to ATP. uncoupling of oxidative Phosphorylation in brown fat mitochondria which involves in heat generation The energy transformation occurring during oxidative phosphorylation may be summarized as follows: Electron transport → energy → proton gradient → ATP synthesis (11)  Clinical correlations : Cyanide poisoning : Cyanide binds to Fe+3 in cytochrome aa3. As a result, O2 can not receive electrons, respiration is inhibited, energy production is halted, and death occurs rapidly.  After cyanide poisoning, the electron transport chain can no longer pump electrons into the intermembrane space. The pH of the intermembrane space would increase, and ATP synthesis would stop.  - Acute myocardial infarction Coronary arteries frequently become narrow because of atherosclerotic plaques. If coronary occlusions occur, regions of heart muscle may be deprived of blood flow and, therefore, of oxygen for prolonged periods of time. Lack of oxygen causes inhibition of the processes of electron transport and oxidative phosphorylation, which results in a decreased production of ATP.  Heart muscle, suffering from a lack of energy required for contraction and maintenance of membrane integrity, becomes damaged. Enzymes from the damaged cells (including the MB fraction of creatine kinase) leak into the blood. If the damage is relatively mild, the person may recover. If heart function is severely compromised, death may result.