Gluconeogenesis. Presented by Dr. Mohammad Saadeh The requirements for the Pharmaceutical Biochemistry II Philadelphia University Faculty of pharmacy

Transcription

1 Gluconeogenesis Presented by Dr. Mohammad Saadeh The requirements for the Pharmaceutical Biochemistry II Philadelphia University Faculty of pharmacy

2 Gluconeogenesis Some tissues, such as the brain, red blood cells, kidney medulla, lens and cornea of the eye, testes, and exercising muscle, require a continuous supply of glucose as a metabolic fuel. Glucose is formed from precursors such as lactate, pyruvate, glycerol and α-keto acids. During an overnight fast, approximately 90% of gluconeogenesis occurs in the liver, with the kidneys providing 10% During prolonged fasting, the kidneys become major glucose producing organs, contributing an estimated 40% of the total glucose production

3 Substrate for gluconeogenesis They include intermediates of glycolysis and the TCA cycle. Glycerol, lactate, and the α-keto acids. 1. Glycerol is released during the hydrolysis of triacylglycerols in adipose tissue. glycerol kinase glycerol phosphate dehydrogenase Glycerol glycerol phosphate dihydroxyacetone phosphate dihydroxyacetone phosphate an intermediate of glycolysis. 2. Lactate is released into the blood by exercising skeletal muscle, and by cells that lack mitochondria, such as RBCs. In the Cori cycle, bloodborne glucose is converted by exercising muscle to lactate, which diffuses into the blood. This lactate is taken up by the liver and reconverted to glucose, which is released back into the circulation.

4 Reactions unique to gluconeogenesis Seven glycolytic reactions are reversible and are used in the synthesis of glucose from lactate or pyruvate. Three of the reactions are irreversible: 1. the conversion of glucose to glucose-6-phospate (by hexokinase), 2. the conversion of fructose 6- phosphate to fructose 1,6- bisphosphate (phosphofructkinase) and 3. the conversion of phosphoenolpyruvate to pyruvate (by pyruvate kinase). These reactions, unique to gluconeogenesis, are described below.

5 Reactions unique to gluconeogenesis A. Carboxylation of pyruvate (need ATP & Biotin) The first step of gluconeogenesis is pyruvate carboxylated by pyruvate carboxylase to oxaloacetate (OAA). Pyruvate carboxylase requires biotin (coenzyme). Hydrolysis of ATP drives the formation of an enzyme biotin CO2 intermediate. This highenergy complex subsequently carboxylates pyruvate to form OAA. Allosteric regulation of Pyruvate carboxylase: 1. activated by high level of acetyl CoA. during fasting, when OAA is used for the synthesis of glucose by gluconeogenesis in the liver and kidney. 2. Inactivated by low levels of acetyl CoA, and pyruvate is primarily oxidized by the pyruvate dehydrogenase complex to produce acetyl CoA that can be further oxidized by the TCA cycle. B. Transport of oxaloacetate to the cytosol Reduction of OAA to malate by mitochondrial malate dehydrogenase (MD) for transfer to the cytosol where it is reoxidized to OAA by cytosolic malate dehydrogenase MD. OAA then converted to PEP by the action of PEP-carboxykinase.

6 Reactions unique to gluconeogenesis C. Decarboxylation of cytosolic oxaloacetate (need GTP) Oxaloacetate is decarboxylated and phosphorylated to PEP in the cytosol by PEP- carboxykinase. The reaction is driven by hydrolysis of guanosine triphosphate (GTP). PEP is acted by the reactions of glycolysis running in the reverse direction until it becomes fructose 1,6-bisphosphate.

7

8 Reactions unique to gluconeogenesis D. Dephosphorylation of fructose 1,6-bisphosphate (regulatory site) Hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate by fructose 1,6 bisphosphatase bypasses the irreversible phosphofructokinase-1 reaction. This reaction is an important regulatory site of gluconeogenesis. Regulation by energy levels within the cell:(see figure )مهم ) slide in the next Fructose 1,6-bisphosphatase: (1). inhibited by elevated levels of adenosine monophosphate (AMP), which is mean energy-poor in cell. (2). Inhibited by fructose 2,6-bisphosphate (see figure 10.5 in the next slide. So inhibited gluconeogenesis. Fructose 1,6-bisphosphatase inhibited by: High levels of ATP & low level of fructose 2,6-bisphosphate and low levels of AMP & high level of fructose 2,6-bisphosphate stimulate gluconeogenesis. High glucagon/insulin ratio causes elevated camp lead to stimulate gluconeogenesis.

9 Figure 10.5: Effect of elevated glucagon on the intracellular concentration of fructose 2,6- bisphosphate in the liver. PFK-2 = phosphofructokinase-2; FBP-2 = fructose bisphosphatase-2.

10 Reactions unique to gluconeogenesis E. Dephosphorylation of glucose 6-phosphate Hydrolysis of glucose 6-phosphate by glucose 6- phosphatase bypasses the irreversible hexokinase reaction, and provides an energetically favorable pathway for the formation of free glucose. Liver and kidney are the only organs that release free glucose from glucose 6-phosphate. This process actually requires two proteins: 1. glucose 6-phosphate translocase, which transports glucose 6-phosphate across the endoplasmic reticulum (ER) membrane. 2. ER enzyme, glucose 6-phosphatase (found only in gluconeogenic cells), which removes the phosphate, producing free glucose.

11 Summary of the reactions of glycolysis and gluconeogenesis Of the 11 reactions required to convert pyruvate to free glucose seven are catalyzed by reversible glycolytic enzymes. The irreversible reactions of glycolysis catalyzed by hexokinase, phosphofructokinase-1, and pyruvate kinase are circumvented by glucose 6- phosphatase, fructose 1,6-bisphosphatase, and pyruvate carboxylase/pepcarboxykinase.

12 Energy consumed by gluconeogenesis To produce 1 molecule of glucose from 2 molecule of pyruvate need: 4 ATP 2 GTP = 2 ATP 2 NADH = 6 ATP Total= (4 ATP+2GTP+2NADH)=12ATP NOTE:

13 Regulation of gluconeogenesis by: A. Glucagon B. Substrate availability A. Glucagon: is hormone release from α cells of pancreatic islets stimulates gluconeogenesis by three mechanisms. 1. Changes in allosteric effectors: Glucagon cause lowers the level of fructose 2,6-bisphosphate. resulting in activation of fructose 1,6-bisphosphatase and inhibition of phosphofructokinase-1, stimulating gluconeogenesis and inhibit glycolysis. 2. Covalent modification of enzyme activity: Glucagon binds its G protein receptor and increase level of cyclic AMP (camp) lead to inactivated pyruvate kinase (phosphorylated) form. This decreases the conversion of PEP to pyruvate that is mean stimulating gluconeogenesis and inhibit glycolysis. 3. Induction of enzyme synthesis: Glucagon increases the transcription of the gene for PEP-carboxykinase, thereby increasing the availability of this enzyme as levels of its substrate rise during fasting. stimulating gluconeogenesis and inhibit glycolysis.

14 Regulation of gluconeogenesis by: B. Substrate availability glucogenic amino acids, influences the rate of hepatic glucose synthesis. Decreased levels of insulin favor mobilization of amino acids from muscle protein, and provide the carbon skeletons for gluconeogenesis. ATP and NADH, coenzymes-cosubstrates required for gluconeogenesis, are primarily provided by the catabolism of fatty acids. C. Allosteric activation by acetyl CoA Allosteric activation of hepatic pyruvate carboxylase by acetyl CoA occurs during fasting. As a result of increased lipolysis in adipose tissue, the liver is flooded with fatty acids. Acetyl CoA accumulates and leads to activation of pyruvate carboxylase. Acetyl CoA inhibits pyruvate dehydrogenase by activating PDH kinase. this single compound can divert pyruvate toward gluconeogenesis and away from the TCA cycle. D. Allosteric inhibition by AMP Fructose 1,6-bisphosphatase is inhibited by AMP that activates phosphofructokinase-1. This results in a reciprocal regulation of glycolysis and gluconeogenesis. Elevated AMP stimulates pathways that oxidize nutrients to provide energy for the cell.

15

16 Tricarboxylic Acid Cycle and Pyruvate Dehydrogenase Complex Presented by Dr. Mohammad Saadeh The requirements for the Pharmaceutical Biochemistry II Philadelphia University Faculty of pharmacy

17 Overview of Tricarboxylic Acid Cycle (TCA cycle)(occurs in mitochondrial) TCA cycle also called Krebs cycle or citric acid cycle plays several roles in metabolism The TCA cycle is an aerobic pathway, because O2 is required as final e- acceptor The cycle also participates in a number of synthetic reactions such as glucose and provides building blocks for synthesis of some amino acids & heme. The citric acid cycle is a metabolic pathway that accomplishes final steps in the breakdown of glucose,amino acids & fatty acids to carbon dioxide (CO 2 ) and water. It take place in the mitochondrial of all eukaryotes, plants, animals, and fungi as well as in some prokaryotes. In the first step of the cycle, oxaloacetate, a 4-carbon acid with two COOH groups, react with acetyl coenzyme A to form citrate. The acetyl CoA comes from the oxidation of pyruvate produced by glycolysis. In each turn of the citric acid cycle, an ATP equivalent is generated in the form of GTP, and several electrons are stored in the form of reduced coenzymes.

18 Reaction of the cycle In the TCA cycle, oxaloacetate is first condensed with an acetyl group from acetyl coenzyme A (CoA), and then is regenerated as the cycle is completed.

19 Reaction of the TCA cycle A. Oxidative decarboxylation of pyruvate: (need 3 enzymes and 5 coenzymes) See Figure 9.2 in the next slide. Pyruvate comes from glycolysis cross the inner mitochondrial membrane to matrix, pyruvate is converted by oxidative decarboxylation to acetyl CoA by pyruvate dehydrogenase. 1. Component enzymes and Coenzymes (See Figure 9.2 in the next slide): pyruvate dehydrogenase multienzyme complex (PDH) (not part of the TCA cycle ): it is included three enzymes and five coenzymes. pyruvate dehydrogenase (PDH or E1, also called a decarboxylase) requires thiamine pyrophosphate (TPP). dihydrolipoyl transacetylase (E2), requires lipoic acid and CoA. dihydrolipoyl dehydrogenase (E3), requires flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NAD + ).

20 Figure 9.2: Mechanism of action of the pyruvate dehydrogenase complex.

21 Reaction of the TCA cycle A. Oxidative decarboxylation of pyruvate: (need 3 enzymes and 5 coenzymes) 2. Regulation of pyruvate dehydrogenase complex 1.Phosphorylation lead to inactive of E1 (PDH) by PDH kinase. 2.Dephosphorylates lead to activate E1 (PDH) by PDH phosphatase. Allosteric regulation of PDH kinase and PDH phosphatase: 1. PDH kinase (cause inactive E1) PDH kinase activated by ATP, acetyl CoA, and NADH. There fore, in the presence of these highenergy signals, the PDH complex is turned off. PDH kinase inhibit by pyruvate. 2. PDH phosphatase (cause stimulating E1) Calcium is a strong activator of PDH phosphatase that is important in skeletal muscle, where release of Ca2+ during contraction stimulates the PDH complex, and thereby energy production.

22 Reaction of the TCA cycle Pyruvate dehydrogenase complex deficiency: A deficiency in the E1 component of the PDH complex cause of congenital lactic acidosis. PDH complex deficiency results in an inability to convert pyruvate to acetyl CoA, causing pyruvate to be shunted to lactic acid via lactate dehydrogenase. This causes particular problems for the brain (X-linked dominant). Note: Arsenic poisoning causes inactivation of PDH complex by binding to lipoic acid.

23 Reaction of the TCA cycle B. Synthesis of citrate from acetyl CoA and oxaloacetate The condensation of acetyl CoA and oxaloacetate to form citrate by citrate synthase (Figure 9.4). citrate synthase is not an allosteric enzyme. It is inhibited by its product (citrate). oxaloacetate causes a conformational change in citrate synthase that generates a binding site for acetyl CoA. Citrate also inhibits phosphofructokinase-1 (PFK-1), the ratelimiting enzyme of glycolysis, and activates acetyl CoA carboxylase (the rate-limiting enzyme of fatty acid synthesis.

24 Reaction of the TCA cycle (مهم) C. Isomerization of citrate Citrate is isomerized to isocitrate by aconitase, an Fe-S protein. D. Oxidation and decarboxylation of isocitrate Isocitrate dehydrogenase catalyzes the irreversible oxidative decarboxylation of isocitrate yielding one NADH and CO 2. This is one of the rate-limiting steps of the TCA cycle. The enzyme is allosterically activated by ADP (a low-energy signal) and Ca 2+. inhibited by ATP and NADH. elevated when the cell has abundant energy.

25 E. Oxidative decarboxylation of α- ketoglutarate Reaction of the TCA cycle The conversion of α-ketoglutarate to succinyl CoA is catalyzed by the α- ketoglutarate dehydrogenase complex. (like PDH complex need coenzyme but not regulated by phosphorylation/dephosphorylation reactions as described for PDH complex). The reaction releases the second CO 2 and produces the second NADH of the cycle. α-ketoglutarate dehydrogenase complex is inhibited by its products, NADH and succinyl CoA, and activated by Ca 2+. α-ketoglutarate is also produced by the oxidative deamination or transamination of the amino acid, glutamate

26 Reaction of the TCA cycle F. Cleavage of succinyl Coenzyme A Succinyl CoA is cleaved by succinate thiokinase (also called succinyl CoA synthetase), producing succinate and GTP. The generation of GTP by succinate thiokinase is another example of substrate-level phosphorylation. Succinyl CoA is also produced from propionyl CoA derived from the metabolism of fatty acids with an odd number of carbon atoms, and from the metabolism of several amino acids. G. Oxidation of succinate Succinate is oxidized to fumarate by succinate dehydrogenase, producing FADH2 by reduced FAD. Succinate dehydrogenase is the only enzyme of the TCA cycle that is embedded in the inner mitochondrial membrane. As such, it functions as Complex II of the electron transport chain.

27 Reaction of the TCA cycle H. Hydration of fumarate Fumarate is hydrated to malate in reversible reaction by fumarase (also called fumarate hydratase). Note: Fumarate is also produced by the urea cycle, in purine synthesis, and during catabolism of the amino acids, phenylalanine and tyrosine. I. Oxidation of malate Malate is oxidized to oxaloacetate by malate dehydrogenase. This reaction produces the third and final NADH of the cycle. Note: The ΔG 0 of the reaction is positive, but the reaction is by the highly exergonic citrate synthase reaction.

28 Energy produced by the TCA cycle Number of ATP molecules produced from the oxidation of one molecule of acetyl CoA (using both substrate-level and oxidative phosphorylation). Total energy from complete mitochondrial oxidation of one molecule of pyruvate to CO 2 (15ATP): 1. Oxidation pyruvate to acetyl CoA produced: 1NADH =3 ATP. 2. Oxidation of acetyl CoA to CO 2 = (3NADH, 1FADH2, 1GTP) = 12 ATP. Total = 15 ATP

29 Regulation of the TCA cycle In The most important regulated enzymes reactions with highly negative ΔG 0 : 1. Citrate synthase 2. Isocitrate dehydrogenase. 3. α-ketoglutarate dehydrogenase

30

31 References: Biochemistry. Lippincott's Illustrated Reviews. 6 th Edition by, Richard A Harvey, Denise R. Ferrier. Lippincott Williams and Wilkins, a Wolters kluwer business

32 Oxidative Phosphorylation Presented by Dr. Mohammad Saadeh The requirements for the Pharmaceutical Biochemistry II Philadelphia University Faculty of pharmacy

33 Electron Transport Chain (ETC) The electron transport chain is a series of protein complexes (called Complexes I, II, III, IV, and V) found in the inner mitochondrial membrane whose main goal is to shuttle electrons to oxygen and protons to form water and create a proton gradient that can be used to synthesized ATP molecules.

34 Reactions of the electron transport chain The electron transport chain consists of 4 major protein Complexes I, II, III, IV, and V. 1. Complex I: (figure 6.9) included 1. NADH dehydrogenase contain ironsulfur centers. embedded in the inner mitochonderial membrane 2. tightly bound of flavin mononucleotide (FMN) 3. The functions if complex I is to accept (2e - + 2H + ) from NADH to FMNH 2 to iron-sulfur center to (coenzyme Q). This reduces ubiquinone (Co Q)into ubiqinol (CoQH 2 ). Ubiquinol then travel to protein complex III. During this process, pump 4 H + from the matrix into intermembrane space.

35 Figure 6.9 Iron-sulfur center of Complex I. [Note: Complexes II and III also contain iron-sulfur centers.

36 Reactions of the electron transport chain 2. Complex II: included 1. Succinate dehydrogenase 2. Flavin adenine dinucleotide (FAD) 3. Peptide subunit with iron-sulfur centers. Electrons from Succinate dehydrogenase used in citric acid cycle to oxidize succinate to fumarate and form FADH 2. FADH 2 gives it is electrons to iron-sulfur center to CoQ, which then moves to complex III. This complex is not proton pump, it does not move H + across the innermembrane.

37 Reactions of the electron transport chain Note: Coenzyme Q is called ubiquinone CoQ is a small hydrophobic molecule that is dissolved in the inner mitochondrial membrane. It acts as an electron carrier and shuttles electrons from Complex I (NADH dehydrogenase), Complex II (succinate dehydrogenase ) to complex III (cytochrome c). 3. Complex III: is called cytochrome bc1 or (cytochrome reductase). The main functions of cytochrome bc1 (Fe+3 converted to Fe+2) is to transfer electrons from CoQ to another electron carrier called cytochrom bc1 to cytochrome c. 2 H + are pumped from the matrix into the intermembrane space. Note: cytochrome c is located in the intermembrane space, water soluble protein and transfers the electrons between complex III and complex IV.

38 Reactions of the electron transport chain 4. Complex IV: is called cytochrome a + a3 or (cytochrome oxidase). The main functions of complex IV is to accept electron from cytochrome c and used them to reduce O2 and produce water. 4 H + are pumped from the matrix into intermembrane space. Cytochrome oxidase contains copper atoms that are required for this complex reaction to occur. Electrons move from Cu A to Cu B - cytochrome a to a 3 to O 2.

39

40 Site-specific inhibitors of electron transport Inhibition of electron transport inhibits ATP synthesis because these processes are tightly coupled. Inhibit complex I (NADH dehydrogenase), so it inhibits the transfer of electrons from ironsulfur centers in complex I to ubiquinone. Inhibit cytochrome bc1 (complexiii ). So it inhibits transfer electrons from cytochrome bc1 to cytochrome c. Inhibit complex IV cytochrome a + a3 (cytochrome oxidase), So it inhibit transfer electron from cytochrome c and used them to reduce O 2 and produce water. H 2 S

41 Release of free energy during electron transport The electrons can be transferred in different forms as hydride ions (:H - ) to NAD +, as hydrogen atoms (. H) to FMN, CoQ, & FAD or as electrons (e - ) to cytochromes. 1. Redox pairs: Oxidation (loss of e - ) accompanied by reduction (gain of e - ) example: Fig 6.11 The tendency to lose electrons, E ο (standard reduction potential) volts. When E ο of redox pairs negative (reductant agent tend to loss electron). When E ο of redox pairs positive (oxidant agent tend to loss electron). ΔG o is related to ΔE ο : n = number of electrons transferred (1 for a cytochrome, 2 for NADH, FADH2, and coenzyme Q) F = Faraday constant (23.1 kcal/volt. mol) ΔE o = E o of the electron-accepting pair minus the Eo of the electron-donating pair ΔG o = change in the standard free energy

42 Release of free energy during electron transport When E ο of redox pairs negative (reductant agent tend to loss electron). When E ο of redox pairs positive (oxidant agent tend to loss electron). ΔG o is related to ΔE ο : n = number of electrons transferred (1 for a cytochrome, 2 for NADH, FADH2, and coenzyme Q) F = Faraday constant (23.1 kcal/volt. mol) ΔE o = E o of the electron-accepting pair minus the E o of the electron-donating pair. ΔG o = change in the standard free energy

43 Release of free energy during electron transport ΔG o for the phosphorylation of ADP to ATP is +7.3 kcal/mol. The transport of a pair of electrons from NADH to oxygen via the electron transport chain produces kcal. NADH produced 3 ATP. Therefore, to produce 3 ATP from 3ADP and 3Pi (3 x 7.3 = 21.9 kcal/mol). NADH is, P:O ratio (ATP made per O atom reduced) of 3:1. The remaining calories are used for ancillary reactions or released as heat. FADH2 produced 2 ATP. FADH2 is 2:1 because it is started from complex II (Complex I is bypassed).

44 Oxidative phosphorylation (phosphorylation of ADP to ATP) A. Chemiosmotic hypothesis OR (Mitchell hypothesis) explains how the free energy generated by the transport of electrons by the electron transport chain is used to produce ATP from ADP + Pi. Two steps are produced ATP from ADP + Pi: 1. proton pump: Complex I,III and IV pumping proton from matrix to the intermembrane space, this creates an electrical and ph gradient, this proton gradient is intermediate in couples oxidation to phosphorylation. 2. Complex V (ATP synthase): is the multisubunit enzyme ATP synthase It is also called F ο /F 1 -ATPase because it contains a domain spanning the inner membrane (F ο ) and extramembranous domain (F 1 ). The chemiosmotic hypothesis proposes that after H + s pumped to inner mitochndrial membrane, they re-enter the mitochondrial matrix by passing through a proton channel (F ο /F 1 -ATPase) in F ο and at the same time dissipating the ph and electrical gradients. F ο rotation causes conformational changes in β subunit of F 1 domain that allow them to bind ADP + Pi phosphorylate ADP to ATP and releases ATP.

45 4 2 4 Electron transport chain shown coupled to the transport of protons. A total 10 protons are pumped for NADH oxidized. [Note: Protons are not pumped at Complex II.]

46 1. Coupling in oxidative phosphorylation: In normal mitochondria, ATP synthesis is coupled to electron transport through the H + gradient. 2. Oligomycin: Oligomycin is a drug binds to the F o domain of ATP synthase, closing the H + channel and preventing reentry of H + into the matrix so it is preventing phosphorylation of ADP to ATP. Electrons transport stops because of the difficulty of pumping any more H + against the steep gradients. The ability to phosphorylate ADP to ATP is known as respiratory control.

47 3. Uncoupling proteins (UCP): occur in the inner mitochondrial membrane of mammals. UCP (carrier proteins) allow protons to reenter the matrix without energy being captured as ATP The energy is released as heat, this is process called nonshivering thermogenesis. UCP1called thermogenin, is responsible for the heat production in the brown adipocytes of mammals.

48 Synthetic uncouplers uncouple oxidative phosphorylation (uncoupled electron flow) such as 2,4- dinitrophenol, aspirin and other salicylates Cause decrease in the proton gradient across the inner mitochondrial membrane and, hence, impaired ATP synthesis therefore defect in energy capture and the metabolism and electron flow to oxygen is increased. This hypermetabolism will be accompanied by elevated body temperature because the energy in fuels is largely wasted, appearing as heat. This explains the fever that accompanies toxic overdoses of these drugs.

49 Membrane transport systems The inner mitochondrial membrane is impermeable to most charged or hydrophilic substances. 1. ATP-ADP transport: The inner mitochondrial membrane requires specialized carriers to transport ADP and Pi from the cytosol into mitochondria, where ATP can be resynthesized. An adenine nucleotide carrier imports one molecule of ADP from the cytosol into mitochondria, while exporting one ATP from the matrix back into the cytosol (see Figure). phosphate carrier is responsible for transporting Pi from the cytosol into mitochondria.

50 Transport of reducing equivalents NADH produced in cytosol can not enter mitochondrial matrix. Therefore, NADH transported from cytosol into mitochondrial matrix by: 1. Glycerol 3-Phosphate Shuttle. 2. Malate-Aspartate shuttle.

51 Transport of reducing equivalents 1. Glycerol 3-Phosphate Shuttle. (produce 2ATP per NADH (from glycolysis) 2 electrons are transferred from NADH (produced in glycolysis) to dihydroxy acetone phosphate (DHAP) by cytosolic glycerophospate dehydrogenase to produce glycerol 3-phosphate. The glycerol 3-phosphate produced moves into the intermembrane space is oxidized by mitochonderial glycerophospate dehydrogenase and back to DHAP as FAD is reduced to FADH2. Transfer 2e - &2H + from FADH 2 to CoQ of ETC. The glycerophosphate shuttle produced 2 ATPs for each NADH produced in glycolysis.

52 Transport of reducing equivalents 1. Glycerol 3-Phosphate Shuttle.

53 Transport of reducing equivalents 2. Malate-Aspartate shuttle. (produce 3 ATP per NADH (from glycolysis) NADH can not transport in the inner mitochondrial membrane. Therefore, NADH can be oxidized to NAD+ by the cytoplasmic malate dehydrogenase as oxaloacetate is reduced to malate. The malate is transported across the inner membrane, and the mitochondrial malate dehydrogenase oxidizes malate to oxaloacetate as mitochondrial NAD+ is reduced to NADH. This NADH can be oxidized by Complex I of the electron transport chain, generating three ATP through the coupled processes of respiration and oxidative phosphorylation.

54 Transport of reducing equivalents 2. Malate-Aspartate shuttle.

55 Inherited defects in oxidative phosphorylation: polypeptides required for oxidative phosphorylation are coded for by mtdna and synthesized in mitochonderia remaining mitochondrial proteins are synthesized in cytosol & transported into mitochondrial. Mutations in mitochondrial DNA (mtdna) are responsible for some cases of mitochondrial diseases, such as Leber hereditary optic neuropathy. Mitochondria and apoptosis: Cytochrome c leave the intermembrane space to the cytoplasm because the pore in outer mitochondrial membrane and subsequent activation of proteolytic caspases results in apoptotic cell death.

56 Science Should be as simple as possible, but not simpler. Albert Einstein

57 References: Biochemistry. Lippincott's Illustrated Reviews. 6 th Edition by, Richard A Harvey, Denise R. Ferrier. Lippincott Williams and Wilkins, a Wolters kluwer business