Protein Metabolism PDF

PROTEIN METABOLISM 1/10/2025 By : Kibrom G. 1 1/10/2025 By : Kibrom G. 2  Dietary proteins are the primary source of the nitrogen that is metabo lized by the body. Amino acids, produced by digestion of dietary proteins, are absorbed through intestinal epithelial cells and en...

PROTEIN METABOLISM 1/10/2025 By : Kibrom G. 1 1/10/2025 By : Kibrom G. 2  Dietary proteins are the primary source of the nitrogen that is metabo lized by the body. Amino acids, produced by digestion of dietary proteins, are absorbed through intestinal epithelial cells and enter the blood. Various cells take up these amino acids, which enter the cellular pools.  They are used for the synthesis of proteins and other nitrogen- containing compounds or they are oxidized for energy. 1/10/2025 By : Kibrom G. 3  In addition to serving as the precursors for the nitrogen-containing compounds of the body and as the building blocks for protein synthesis, amino acids are also a source of energy. Amino acids are directly oxidized or they are converted to glucose and then oxidized or stored as glycogen. They also may be converted to fatty acids and stored as adipose triacylglycerols. Glycogen and triacylglycerols are oxidized during periods of fasting. The liver is the major site of amino acid oxidation. However, most tissues can oxidize the branched-chain amino acids (leucine, isoleucine, and valine). 1/10/2025 By : Kibrom G. 4  Before the carbon skeletons of amino acids are oxidized, the nitrogen must be removed. Amino acid nitrogen forms ammonia, which is toxic to the body. In the liver, ammonia and the amino groups from amino acids are converted to urea, which is nontoxic, water-soluble, and readily excreted in the urine. The process by which urea is produced is known as the urea cycle. The liver is the organ responsible for producing urea. Branched-chain amino acids can be oxidized in many tissues, but the nitrogen must always travel to the liver for disposal. 1/10/2025 By : Kibrom G. 5 1/10/2025 By : Kibrom G. 6 1/10/2025 By : Kibrom G. 7 Protein Digestion and Amino Acid Absorption Proteolytic enzymes (also called proteases) break down dietary proteins into their constituent amino acids in the stomach and the intestine. Many of these digestive proteases are synthesized as larger, inactive forms known as zymogens. After zymogens are secreted into the digestive tract, they are cleaved to produce the active proteases. 1/10/2025 By : Kibrom G. 8  In the stomach, pepsin begins the digestion of proteins by hydrolyzing them to smaller polypeptides. The contents of the stomach pass into the small intestine, where enzymes produced by the exocrine pancreas act. The pancreatic proteases (trypsin, chymotrypsin, elastase, and the carboxypeptidases) cleave the polypeptides into oligopeptides and amino acids. 1/10/2025 By : Kibrom G. 9  Further cleavage of the oligopeptides to amino acids is accomplished by enzymes produced by the intestinal epithelial cells. These enzymes include aminopeptidases located on the brush border and other peptidases located within the cells. Ultimately, the amino acids produced by protein digestion are absorbed through the intestinal epithelial cells and enter the blood. A large number of overlapping transport systems exist for amino acids in cells. 1/10/2025 By : Kibrom G. 10  Some systems contain facilitative transporters, whereas others express sodium- linked transporters, which allow the active transport of amino acids into cells. Defects in amino acid transport can lead to disease. Proteins are also continually synthesized and degraded (turnover) in cells. A wide variety of proteases exist in cells to carry out this activity. 1/10/2025 By : Kibrom G. 11  Lysosomal proteases (cathepsins) degrade proteins that enter lysosomes. Cytoplasmic proteins targeted for turnover are covalently linked to the small protein ubiquitin, which then interacts with a large protein complex, the proteasome, to degrade the protein in an adenosine triphosphate (ATP)-dependent process. The amino acids released from proteins during turnover can then be used for the synthesis of new proteins or for energy generation. 1/10/2025 By : Kibrom G. 12 1/10/2025 By : Kibrom G. 13  PROTEIN DIGESTION 1/10/2025 By : Kibrom G. 14 PROTEIN DIGESTION The digestion of proteins begins in the stomach and is completed in the intestine. The enzymes that digest proteins are produced as inactive precursors (zymogens) that are larger than the active enzymes. The inactive zymogens are secreted from the cells in which they are synthesized and enter the lumen of the digestive tract, where they are cleaved to smaller forms that have proteolytic activity. 1/10/2025 By : Kibrom G. 15  These active enzymes have different specificities; no single enzyme can completely digest a protein. However, by acting in concert, they can digest dietary proteins to amino acids and small peptides, which are cleaved by peptidases associated with intestinal epithelial cells 1/10/2025 By : Kibrom G. 16 A. Digestion of Proteins in the Stomach Pepsinogen is secreted by the chief cells of the stomach. The gastric parietal cells secrete HCl. The acid in the stomach lumen alters the conformation of pepsinogen so that it can cleave itself, producing the active protease pepsin. Thus, the activation of pepsinogen is autocatalytic. Dietary proteins are denatured by the acid in the stomach. 1/10/2025 By : Kibrom G. 17  This serves to inactivate the proteins and partially unfolds them such that they are better substrates for proteases. However, at the low pH of the stomach, pepsin is not denatured and acts as an endopeptidase, cleaving peptide bonds at various points within the protein chain. Although pepsin has a fairly broad specificity, it tends to cleave peptide bonds in which the carboxyl group is provided by an aromatic or acidic amino acid. Smaller peptides and some free amino acids are produced. 1/10/2025 By : Kibrom G. 18 1/10/2025 By : Kibrom G. 19 B. Digestion of Proteins by Enzymes from the Pancreas As the gastric contents empty into the intestine, they encounter the secretions from the exocrine pancreas. One of these secretions is bicarbonate, which, in addition to neutralizing the stomach acid, raises the pH such that the pancreatic proteases, which are also present in pancreatic secretions, can be active. As secreted, these pancreatic proteases are in the inactive proenzyme form (zymogens). 1/10/2025 By : Kibrom G. 20  Because the active forms of these enzymes can digest each other, it is important for their zymogen forms all to be activated within a short span of time. This feat is accomplished by the cleavage of trypsinogen to the active enzyme trypsin, which then cleaves the other pancreatic zymogens, producing their active forms. 1/10/2025 By : Kibrom G. 21  The zymogen trypsinogen is cleaved to form trypsin by enteropeptidase (a protease, formerly called enterokinase) secreted by the brush-border cells of the small intestine. Trypsin catalyzes the cleavages that convert chymotrypsinogen to the active enzyme chymotrypsin, proelastase to elastase, and the procarboxypeptidases to the carboxypeptidases. Thus, trypsin plays a central role in digestion because it both cleaves dietary proteins and activates other digestive proteases produced by the pancreas. 1/10/2025 By : Kibrom G. 22  Trypsin, chymotrypsin, and elastase are serine proteases that act as endopeptidases. Trypsin is the most specific of these enzymes, cleaving peptide bonds in which the carboxyl (carbonyl) group is provided by lysine or arginine. Chymotrypsin is less specific but favors residues that contain hydrophobic or acidic amino acids. Elastase cleaves not only elastin but also other proteins at bonds in which the carboxyl group is contributed by amino acid residues with small side chains (alanine, glycine, or serine). The actions of these pancreatic endopeptidases continue the digestion of dietary proteins begun by pepsin in the stomach. 1/10/2025 By : Kibrom G. 23  The smaller peptides formed by the action of trypsin, chymotrypsin, and elastase are attacked by exopeptidases, which are proteases that cleave one amino acid at a time from the end of the chain. Procarboxypeptidases, zymogens produced by the pancreas, are converted by trypsin to the active carboxypeptidases. These exopeptidases remove amino acids from the carboxyl ends of peptide chains. Carboxypeptidase A preferentially releases hydrophobic amino acids, whereas carboxypeptidase B releases basic amino acids (arginine and lysine). 1/10/2025 By : Kibrom G. 24  Pancreatic enzymes 1/10/2025 By : Kibrom G. 25 1/10/2025 By : Kibrom G. 26 1/10/2025 By : Kibrom G. 27  Digestion of Proteins by Enzymes from Intestinal Cells Exopeptidases produced by intestinal epithelial cells act within the brush border and also within the cell. Aminopeptidases, located on the brush border, cleave one amino acid at a time from the amino end of peptides. Intracellular peptidases act on small peptides that are absorbed by the cells. 1/10/2025 By : Kibrom G. 28  The concerted action of the proteolytic enzymes produced by cells of the stomach, pancreas, and intestine cleaves dietary proteins to amino acids. The digestive enzymes digest themselves as well as dietary protein. They also digest the intestinal cells that are regularly sloughed off into the lumen. These cells are replaced by cells that mature from precursor cells in the duodenal crypts. The amount of protein that is digested and absorbed each day from digestive juices and cells released into the intestinal lumen may be equal to, or greater than, the amount of protein consumed in the diet (50–100 g) 1/10/2025 By : Kibrom G. 29 ABSORPTION OF AMINO ACIDS Amino acids are absorbed from the intestinal lumen through secondary active Na+- dependent transport systems and through facilitated diffusion. 1/10/2025 By : Kibrom G. 30 Cotransport of Na+ and Amino Acids Amino acids are absorbed from the lumen of the small intestine principally by semi specific Na+-dependent transport proteins in the luminal membrane of the intestinal cell brush border, similar to that of carbohydrate transport. The cotransport of Na+ and the amino acid from the outside of the apical membrane to the inside of the cell is driven by the low intracellular Na+ concentration. Low intracellular Na+ results from the pumping of Na+ out of the cell by a Na+,K+-ATPase on the serosal membrane. 1/10/2025 By : Kibrom G. 31 1/10/2025 By : Kibrom G. 32 1/10/2025 By : Kibrom G. 33 1/10/2025 By : Kibrom G. 34 1/10/2025 By : Kibrom G. 35 PROTEIN TURNOVER AND REPLENISHMENT OF THE INTRACELLULAR AMINO ACID POOL The amino acid pool within cells is generated both from dietary amino acids and from the degradation of existing proteins within the cell. All proteins within cells have a half-life (t1/2), a time at which 50% of the protein that was synthesized at a particular time will have been degraded. Mechanisms for intracellular degradation of unnecessary or damaged proteins involve lysosomes and the ubiquitin/proteasome system. 1/10/2025 By : Kibrom G. 36 1. Lysosomal Protein Turnover Lysosomes participate in the process of autophagy, in which intracellular components are surrounded by membranes that fuse with lysosomes, and endocytosis. Autophagy is a complex regulated process in which cytoplasm is sequestered into vesicles and delivered to the lysosomes. Within the lysosomes, the cathepsin family of proteases degrades the ingested proteins to individual amino acids. 1/10/2025 By : Kibrom G. 37  The recycled amino acids can then leave the lysosome and rejoin the intracellular amino acid pool. starvation of a cell is a trigger to induce this process.  This will allow old proteins to be recycled and the newly released amino acids used for new protein synthesis, to enable the cell to survive starvation conditions. 1/10/2025 By : Kibrom G. 38 The Ubiquitin-Proteasome Pathway Ubiquitin is a small protein (76 amino acids) that is highly conserved. Its amino acid sequence in yeast and humans differs by only three residues. Ubiquitin targets intracellular proteins for degradation by covalently binding to the ϵ-amino group of lysine residues. This is accomplished by a three-enzyme system that adds ubiquitin to proteins targeted for degradation. 1/10/2025 By : Kibrom G. 39  Oftentimes, the target protein is polyubiquitinylated, in which additional ubiquitin molecules are added to previous ubiquitin molecules, forming a long ubiquitin tail on the target protein. After polyubiquitinylation is complete, the targeted protein is released from the three enzyme complex. A protease complex, known as the proteasome, then degrades the targeted protein, releasing intact ubiquitin that can again mark other proteins for degradation. 1/10/2025 By : Kibrom G. 40  The basic proteasome is a cylindrical 20S protein complex with multiple internal proteolytic sites.  ATP hydrolysis is used both to unfold the tagged protein and to push the protein into the core of the cylinder. The complex is regulated by cap protein complexes, which bind the ubiquinylated protein (a step that requires ATP) and deliver them to the complex. 1/10/2025 By : Kibrom G. 41  After the target protein is degraded, the ubiquitin is released intact and recycled. The resultant amino acids join the intracellular pool of free amino acids. 1/10/2025 By : Kibrom G. 42  METABOLIC FATES OF AMINO ACIDS Body protein biosynthesis. Small peptide biosynthesis (GSH). Synthesis of non protein nitrogenous (NPN) compounds (creatine, urea,ammoniaanduricacid) Deamination &Transamination to synthesized a new amino acid or glucose or ketone bodies or produce energy in starvation.  Metabolism of Amino Acids: 1.Removal of ammonia by : Deamination Oxidative deamination Glutamate dehydrogenase in mitochondria Amino acid oxidase in peroxisomes Direct deamination (non oxidative) Dea. by dehydration (-H2O) Dea. by desulhydration(-H2S)  Transamination (GPT & GOT)  transdeamination. 2.Fate of carbon-skeletons of amino acids 3.Metabolism of ammonia  Deamination of Amino Acids Deamination of Amino Acids A. Oxidative Deamination: 1. Glutamate dehydrogenase , mitochondrial , potent, major deaminase  It is allosterically stimulated by ADP & inhibited by ATP, GTP & NADH.  Thus, high ADP (low caloric intake) increases protein degradation high ATP ( well fed-state) decreases deamination of amino acids & increases protein synthesis.  2. Amino Acid Oxidases  The minor pathway for deamination of amino acids.  They are found in peroxisomes of liver and kidney.  L-amino acid oxidases utilize FMN while D-a.a. oxidases utilize FAD.  D-amino acid oxidases are highly active than L- amino acid oxidases especially in kidney and liver due to: the function of D-amino acid oxidases is the rapid and irreversible break down of D-amino acids since:  D-amino acids are potent inhibitors to L-amino acids oxidases  B. Non-oxidative deamination: (Direct Deamination ) 1) Deamination by dehydration: Serine & Threonine 2. Deamination by desulfhydration : (cysteine)  Transamination Aminotransferases are active both in cytoplasm and mitochondria e.g.: 1. Aspartate aminotransferase (AST), Glutamate oxaloacetate transaminase (GOT), 2. Alanine aminotransferase (ALT), Glutamate pyruvate transaminase, (GPT) In all transamination reactions, α-ketoglutarate (α –KG) acts as amino group acceptor. Most, but not all amino acids undergo transamination reaction with few exceptions (lysine, threonine and imino acids)  The role of PLP as Co-aminotransferase  PLP binds to the enzyme via schiff’s base & ionic salt bridge & helps in transfer of amino group between amino acid and keto acid (KG):  Metabolic Significance of Transamination Reactions It is an exchange of amino nitrogen between the molecules with out a net loss. This metabolically important because:  There is no mechanism for storage of a protein or amino acids.  In case of low energy (caloric shortage),the organism depends on oxidation of the keto acids derived from transamination of amino acids.  It is important for formation of the non-essential amino acids  Transdeamination  Due to…L-amino acid oxidases, but not glutamate dehydrogenase, can sluggish (decrease) the rate of deamination of the amino acids.  So… the most important and rapid way to deamination of amino acids is first transamination with α-ketoglutarate followed by deamination of glutamate. Therefore glutamate through transdeamination serves to a funnel ammonia from all amino acids.  THE FATE OF CARBON-SKELETONS OF AMINO ACIDS a)Simple degradation: (amino acid Common metabolic intermediate) Alanine Pyruvate Glutamate α -ketoglutarate Aspartate Oxaloacetate b) Complex degradation: (amino acid---Keto acid-----complex pathway----Common metabolic intermediate)  Amino acids whose keto acids are metabolized via more complex path way e.g. Tyrosine, Lysine, Tryptophan c) Conversion of one amino acid into another amino acid before degradation: Phenylalanine is converted to tyrosine prior to its further degradation The common metabolic intermediates that raised from the degradations of amino acids are: acetyl CoA, pyruvate, one of the krebs cycle intermediates (α-ketoglutarate, succinyl CoA, fumarate& oxaloacetate)  Metabolism of the Common Intermediates  Oxidation: all amino acids can be oxidized in TCA cycle with energy production Fatty acids synthesis: some amino acids provide acetyl CoA e.g. leucine and lysine (ketogenic amino acids). Gluconeogenesis: keto acids derived from amino acids are used for synthesis of glucose(is important in starvation). Glucogenic Ketogenic Glucogenic& Ketogenic Ala,Ser,Gly,Cys Leu ,Lys Phe,Tyr,Trp,Ile,Thr Arg,His,Pro,Glu, Gln,Val,Met,Asp,Asn.  METABOLISM OF AMMONIA Ammonia is formed in body from  From amino acids: 1.Transdeamination in liver 2.amino acid oxidases and amino acid deaminases in liver and kidney.  Deamination of physiological amines: by monoamine oxidase (histamine, adrenaline, dopamine and serotonine).  Deamination of purine nucleotides: especially adenine nucleotides  From bacterial action in the intestine on dietary protein & on urea in the gut.  NH3 is also produced by glutaminase on glutamine.  Metabolic Disposal of Ammonia Excess ammonia is toxic to CNS, it is fixed into nontoxic metabolite for reuse or excretion according to the body needs:  a. Formation of Glutamate:  b. Glutamine Formation: Muscle, brain  Glutamine is storehouse of ammonia & transporter form of ammonia. In brain, glutamine is the major mechanism for removal of ammonia while in liver is urea formation...Circulating glutamine is removed by kidney, liver and intestine where it is deamidated by glutaminase.  c) Urea formation This reaction is important to kidney due to kidney excretes NH4+ ion to keep extracellular Na+ ion in body and to maintain the acid-base balance.  c. Urea Formation Urea is the principal end-product of protein metabolism in humans.  It is important route for detoxication of NH3.  It is operated in liver, released into blood and cleared by kidney. Urea is highly soluble, nontoxic and has a high nitrogen content(46%), so…it represents about 80- 90% of the nitrogen excreted in urine per day in man.  Biosynthesis of urea in man is an energy-requiring process.  It takes place partially in mitochondria and partially in cytoplasm. The Urea Cycle (The Ornithine Cycle, Kreb's Henseleit Cycle)  Urea Cycle Connect s t o TCA Cycle H (-)O CCH C CO(-) 2 2 2 Ornithine Citrulline NH2 Aspartate Urea Urea Cycle Arginine Argininosuccinate Oxaloacetate Malate H (-)OC C C 2 H CO( 2 -) TCA Cycle Citrate Fumarate -Ketoglutarate  Metabolic Significant Aspects of Urea Cycle A. Energy Cost: Energy cost of the cycle is only one ATP. B. urea cycle is related to TCA cycle: 1.CO2 2.Aspartate arises via transamination of OAA with glutamate. Thus, depletion of OAA will decrease urea formation (as in malonate poisoning). 3.Fumarate enters TCA cycle C. Sources of Nitrogen in urea:freeNH3 and aspartate. N.B.glutamate is the immediate source of both NH3(via oxidative deamination by Glu.Dehyd.) and aspartate nitrogen(through transamination of oxaloacetate byAST).  Importance of Urea Cycle 1.Formation of arginine (in organisms synthesizing arginine) & formation of urea(in ureotelic organisms, man)due to presence of arginase. 2.Liver shows much higher activity of arginase than brain or kidney for formation of urea while in brain or kidney is the synthesis of arginine. 3.Synthesis of non protein amino acids(ornithine and citrulline) in body.  Regulation of UreaCycle 1) Activity of individual enzymes: THE RATE LIMITING STEPS a) Carbamoyl phosphate synthase-1 b) Ornithine transcarbamyolase. c) Arginase. N-acetylglutamate is activator for carbamoyl phosphate synthase-1  It enhances its affinity for ATP.  It is synthesized from acetyl CoA and glutamate.  Its hepatic concentration increases after intake of a protein diet, leading to an increased rate of urea synthesis. Activity of ornithine transcarbamyolase is limited by the concentration of its co-substrate "ornithine".  2) Regulation of the flux through the cycle a) Flux of ammonia: 1. By amino acids release from muscle (alanine, glutamine) 2. Metabolism of glutamine in the intestine 3. Amino acids degradation in the liver. b) Availability of ornithine. c) Availability of aspartate. since aspartate is required in equimolar amounts with ammonia, this is satisfied by of transdeamination. 3) Change in the level of Enzymes:  Arginase & other urea forming enzymes are adaptive enzymes thus a protein rich diet will increase their biosynthesis rate & the opposite is true for low protein diet.  However, in starvation, where the body is forced to use its own tissue protein as fuel, there is an increase in urea-forming enzymes.  METABOLISM OF INDIVIDUAL AMINO ACIDS 1. Metabolism of Glycine: non essential, glucogenic. Biosynthesis of glycine: Special Functions of Glycine a-Protein, Hormones & enzymes. b- Heme c- Purines d- Creatine and Glutathione f- Conjugating reactions: Glycine + Cholic acid glycocholate. Glycine + Benzoic acid Hippuric acid 1.Formation of Glutathione (GSH) 2. Formation of creatine (Methyl guanidoacetate) Cr-P is the storage form of high energy phosphate in muscle Creatinine is excreted in urine & increases on kidney failure due to its filteration is decreased. Its level is constant per 24 hrs& is proportional to muscle mass in human.  2. Metabolism of Serine Serine : nonessential & glucogenic It is synthesize from glycine or Intermediate of glycolysis All enzymes are activated by testosterone in liver, kidney & prostate.  Degradative Pathways of Serine  Serine is important in synthesis of: a. Phosphoprotein b. Purines & pyrimidine c. Sphingosine d. Choline e. Cysteine  3. Metabolism of Sulfur-Containing amino acids (Methionine, cyteine & Cystine): a Metabolism of methionine: (essential) In transmethylation there are b. Metabolism of Cysteine& Cystine They are interconvertable &They are not essential-can be synthesized from Met & Ser Degredative pathway of cysteine  Biochemical functions of cysteine 1- PAPS Formation: (3'-phosphoadenosine,5‘ phosphosulphate) active sulphate used in formation of sulfate esters of steroids, alcohol, phenol, some lipids, proteins and mucopolysaccharides 2- Sulfur of COASH, GSH, vasopressin, insulin 3-Detoxication reaction of bromo, chloro, iodobenzene, naphthalene and anthracene & of phenol, cresol, indol and skatol that is formed by the action of intestinal bacteria on some amino acids in large intestine with formation of ethereal sulfates which is water soluble and rapidly removed by the kidney 4- Taurine Formation ( with bile acids form taurocholate) 4. Aromatic amino acids A. Metabolism of Phenylalanine : (glucogenic & ketogenic) b. Tyrosine is a precursor of: 1.DOPA (3,4 dihydroxy phenylalanine) 2.Thyroid hormones: Thyroxine Formation  Thyroglobulin(Tgb)  It is the precursor of T3 and T4  It is large, iodinated, glycosylated protein.  It contains 115 tyrosine residues each of which is a potential site of iodination.  70% iodide in Tgb exists in the inactive forms MIT&DIT WHILE  30% is in T3& T4  About 50 μg Thyroglobulin is secreted each day. c. Tryptophan (essential, glucogenic &ketogenic) I.3-hydroxyanthranilic acid pathway: Trp pyrrolase Inc.by Cortico. & tryptophan & Dec.by Niacin, NAD & NADP  2) Serotonin Pathway  Neurotransmitter  Founds in mast cells& platelets.  Vasoconstrictor for bronchioles  Transmitter in GIT to release the peptide hormones.  3) Melatonin formation pathway  It is the hormone of pineal body in brain of man.  Formed by the acetylation and methylation of serotonin.  It has effects on hypothalamic-pituitary system.  It blocks the action of MSH&ACTH.  It is important in regulation of gonad & adrenal functions.  It has a circadian rhythm due to its formation occurs only in dark, due to high activity of N acetyl transferase enzyme so it is a biological clock.  It keeps the integrity of cells during aging due to it has an anti oxidant property  It enhances the body defense against infection in AIDS patients by increasing the number of immune cells.  It reduces the risk of cancer & heart diseases  5. Basic Amino Acids: 1) Histidine (glucogenic amino acid):  Histamine is a chemical messenger that mediates allergic and inflammatory reactions, gastric acid secretion and neurotransmission in the brain.  (2) Arginine: (nonessential & glucogenic amino acid):  It participates in formation of:  a) Creatine b) Polyamines C) Nitric oxide NO (Free radical gas). 3) Lysine: (essential, ketogenic) it is involved in the formation of histone, hydroxy lysine & carnitine: 6. Acidic Amino Acids : 1.Glutamic acid : (nonessential & glucogenic amino acid). It participates in formation of:  1- GSH.  2- Glutamine: as storage and transporter form of ammonia  3- GABA ( -aminobutyric acid) neurotransmitter in brain.  2. Aspartic acid:Acidic, non essential & glucogenic  1.Arginosuccinate in urea cycle. 2.Alanine by decarboxylation. 3. Oxalate & glucose by T.A.  Amino acids as precursors of neurotransmitters  Arginine --------------NO  Tryptophan-----------Serotonin  Histidine--------------Histamine  Tyrosine------dopa,dopamine, NE&E  Glutamic acid--------GABA  Glycolysis,PDHcomplex,krebs cycle,and ETS pathways By :Elias T/mariam (BSc,MSc) 1 Outline of the lecture  Glycolysis  Pyruvate dehydrogenase complex  Krebs cycle  Electron transport chain; oxidative phosphorylation; ATP yields 2  Glycolysis  Essentially all living cells use GLYCOLYSIS as their main pathway for glucose metabolism.  It occurs in the cytosol and is also called the Embden-Meyerhof pathway.  Glycolysis is the conversion of glucose to pyruvate. It is also used for the conversion of glucose to lactate when pyruvate is converted to lactate (e.g. during anaerobic glycolysis).  There are 10 enzyme steps involved. Three of these steps are irreversible.  In the presence of oxygen, pyruvate enters mitochondria and is further metabolized to acetyl- Coenzyme A (acetyl-CoA), which is then metabolized further by the Krebs cycle.  In the absence of oxygen, anaerobic glycolysis occurs and the pyruvate is converted to lactate (catalyzed by lactate dehydrogenase, LDH) in the cytosol.  Glycolysis produces ATP, which is useful for cells to survive low oxygen conditions, where mitochondrial oxidative phosphorylation is slow. However, much less ATP is produced by glycolysis from one glucose molecule than is produced if the pyruvate was further metabolized by the Krebs cycle.  Many cancer cells produce lactate even in the presence of oxygen. This is called aerobic glycolysis. It is clear from more recent research also that many normal cells (for example red blood cells, proliferating T-lymphocytes, brain cells, also produce lactate even in the presence of oxygen, and 3 therefore aerobic glycolysis is not confined to cancer cells.  Glycolysis  The overall pathway of glycolysis is: Glucose + 2 NAD+ + 2 ADP + 2 Pi → 2 Pyruvate + 2 NADH + 2 H+ + 2 ATP + 2 H2O  The first step is the conversion of glucose (which enters cells from the bloodstream/ extracellular environment) to glucose-6-phosphate, catalyzed by hexokinase.This step uses one ATP molecule: Glucose + ATP Glucose-6-phosphate + ADP The phosphorylation prevents the glucose-6-phosphate from leaving the cells and the reaction is irreversible. Hexokinase is an important step in glycolysis. It is a regulatory step for glycolysis; glucose-6- phosphate can be used for various purposes (glycolysis, pentose phosphate pathway, glycogen synthesis, gluconeogenesis). In some cells, e.g., liver, pancreas, there is an important type (isoform) of hexokinase called glucokinase which has a very important function as a blood glucose “sensor.” 4 5  GLYCOLYS 6  Glycolysis  The next step (isomerization) converts glucose-6-phosphate to fructose-6-phosphate. Enzyme: phosphoglucose isomerase.  The fructose-6-phosphate is then converted to fructose-1,6-bisphosphate by phosphofructokinase (PFK): fructose-6-phosphate + ATP fructose-1,6-bisphosphate + ADP This step is also important because it is a major regulatory step in glycolysis. It uses up another ATP, so that by now already the energy from 2 ATP molecules have been used for each glucose molecules metabolized. This step is also irreversible. *The initial steps of glycolysis, therefore, use 2 ATP molecules per glucose molecule and are “investments” to get the pathway to the next stages, 7 where eventually more ATP will be synthesized than used up.  Glycolysis  Fructose-1,6-bisphosphate is then cleaved into two 3-carbon molecules: glyceraldehyde-3-phosphate dehydrogenase and dihydroxyacetone phosphate. The enzyme is aldolase (also called fructose-1,6- bisphosphate aldolase).  The two 3-carbon molecules, dihydroxyacetone phosphate and glyceraldehyde-3-phosphate dehydrogenase, are both triose phosphates, and are isomers of each other. They are interconverted to each other by triose phosphate isomerase. The conversion of dihydroxyacetone phosphate to glyceraldehyde-3-phosphate is favoured, and the glyceraldehyde-3-phosphate is further metabolised by glycolysis.  From this step onwards, there are two 3-carbon molecules for each glucose molecule.  The next step is catalyzed by glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and uses NAD+ to oxidize glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate and NADH:  NAD+ + glyceraldehyde-3-phosphate + phosphate 1,3-bisphosphoglycerate + NADH + H+ This is important because it generates NADH, which can be used to make ATP. 8  Glycolysis  The next step, catalyzed by phosphoglycerate kinase, is the first step in which ATP is made. This is an example of substrate-level phosphorylation, in which ATP is made from ADP directly in an enzymatic reaction. This is distinct from the way ATP is produced by oxidative phosphorylation, from reduced molecules (NADH and FADH), via the electron transport chain. 1,3-bisphosphoglycerate + ADP 3-phosphoglycerate + ATP  This means that 2 ATP molecules are made for each glucose molecule at this step, because two 1,3-bisphosphoglycerate molecules are derived from one glucose molecule.  Since 2 ATP molecules were used up in the hexokinase and phosphofructokinase steps, the net yield of ATP so far is zero (2 ATP used, 2 ATP made). 9  Glycolysis  The 3-phosphoglycerate is then isomerized to 2-phosphoglycerate by the enzyme, phosphoglycerate mutase.  Following this, 2-phosphoglycerate is converted to phosphoenolpyruvate (PEP). The enzyme is enolase. Phosphoenolpyruvate contains a high energy enol-phosphate linkage, which is even more high in energy than the terminal phosphodiester bond in ATP. In the next step, this energy from the phosphoenol group of PEP is transferred to ADP to make ATP.  PEP is then converted to pyruvate, the final product of glycolysis, in the presence of oxygen. In anaerobic conditions, lactate is the final product of glycolysis. The phosphate group of the phosphoenolpyruvate is transferred in this reaction, catalyzed by pyruvate kinase, to ADP to form another ATP molecule: phosphoenolpyruvate + ADP pyruvate + ATP This is another example of substrate-level phosphorylation, where ATP is made at the reaction level and not via reducing equivalents through oxidative phosphorylation. This step makes another 2 ATP molecules per original glucose molecule. This now means that a net of 2 ATP molecules are made from one glucose molecule by glycolysis. 10  Glycolysis  Under low oxygen (anaerobic) conditions, the pyruvate cannot enter mitochondria and enter the Krebs cycle and undergo oxidative phosphorylation, because this process needs oxygen.  Therefore, the pyruvate is converted to lactate in the cytosol by the enzyme, lactate dehydrogenase (LDH). This involves reducing pyruvate to lactate using NADH. This step uses the NADH generated in an earlier step (glyceraldehyde-3-phosphate dehydrogenase step) to regenerate NAD+ and maintain a stable redox state of the cytoplasm. 11  Glycolysis  In the presence of enough oxygen, the pyruvate formed in the cytosol during glycolysis can enter mitochondria and be used, via the Krebs cycle and oxidative phosphorylation, to make ATP.  The net yield of ATP from glycolysis during aerobic conditions is higher that 2 ATP molecules per glucose because the reducing equivalents from the NADH generated during glycolysis (at the glyceraldehyde-3-phosphate step) can be used to make ATP via oxidative phosphorylation. This will be discussed in the next lecture.  When lactate is the product of glycolysis, the NADH produced at the glyceraldehyde dehydrogenase step is used up in the conversion of pyruvate to lactate (by lactate dehydrogenase). This regenerates NAD+ for re-use. In this case, therefore, NADH does not enter mitochondria for ATP production. So, glycolysis that produces lactate produces a net of only 2 ATP molecules.  Some tissues, for example skeletal muscle, may use anaerobic glycolysis on occasions when the demand for ATP exceeds the capacity of mitochondrial ATP production to supply ATP (due to limited supply of oxygen, for example during intense exercise).  Red blood cells lack mitochondria and so produce lactate from glycolysis. Other tissues, for example brain, also produce lactate normally. We will return to these issues later and discuss the idea that the classical idea of cytosolic pyruvate being the end product of glycolysis and the source of mitochondrial acetyl-CoA might need to be revised. 12 Glyceraldehyde-3-phosphate dehydrogenase step 13  Glycolysis: pyruvate may be converted to lactate (in anaerobic conditions) or enter mitochondria to be converted to acetyl-CoA, which then enters the Krebs cycle (citric acid cycle). 14  Adenylate Kinase  Cells generally maintain high ATP : AMP ratios in order to keep the ATP supply available for metabolic and biological functions. The ATP concentration may be 100 times higher than the AMP concentration.  The ubiquitous enzyme, adenylate kinase, catalyses the following reaction: 2ADP ATP + AMP * Adenylate kinase relays information about the energy status of a cell to other molecular systems, in particular AMP-activated protein kinase (AMPK). 15  Summary of Glycolysis  There are 10 enzyme-catalyzed steps in glycolysis from glucose to pyruvate, and 11 steps from glucose to lactate.  Glycolysis to pyruvate produces a net of 2 ATP molecules per glucose molecule plus 2 NADH molecules per glucose molecule.  The first step (hexokinase) is irreversible and also glucose-6-phosphate cannot leave the cell, because phosphorylated molecules do not diffuse out of cells well. This is true for all intermediates except pyruvate- they are all phosphorylated!  The first phase of glycolysis actually uses up ATP (2 ATP per glucose molecule), rather than makes ATP. This phase “sets up” the pathway for subsequent net ATP production in the later steps of glycolysis.  Know the steps where ATP is used and where ATP is made.  Know the step (glyceraldehyde-3-phosphate dehydrogenase) where NADH is formed.  Glycolysis to lactate produces a net of 2 ATP molecules per glucose molecule and ZERO NADH, because the NADH is used to convert pyruvate to lactate (lactate dehydrogenase step).  One of the main functions of glycolysis is to produce energy in the form of ATP phosphodiester bonds from energy stored in the glucose molecule. Another function is to produce pyruvate for further ATP production in mitochondria.  The phosphofructokinase step (fructose-6-phosphate to fructose-1,6-bisphosphate) is the step in glycolysis that commits the pathway to glycolysis, since the first irreversible step (hexokinase) produces glucose-6-phosphate, which can be diverted to other pathways (e.g. pentose phosphate pathway).  Summary of Glycolysis  The ATPs produced in glycolysis are mad by substrate-level phosphorylation, rather than by oxidative phosphorylation, which occurs in mitochondria via production of reducing equivalents (FADH 2 and NADH) and the electron transport pathway.  Understand: Aerobic respiration (oxidative glycolysis), anaerobic glycolysis, aerobic glycolysis.  There are 3 irreversible steps in glycolysis, where there is a significant negative Gibbs free energy. These are: Hexokinase (HK) Phosphofructokinase (PFK) Pyruvate kinase (PK)  These steps are also rate-limiting (rate-controlling) steps, where flux through the pathway is regulated.  Because glycolysis is irreversible at 3 steps, gluconeogenesis (the production of glucose from precursor molecules such as pyruvate, glycerol, alanine, lactate and others) cannot simply be a reversal of glycolysis: different enzymes catalyse the conversions in the in the opposite direction at these irreversible steps. (e.g. hexokinase step is irreversible, so a different enzyme (glucose-6-phosphatase) converts glucose-6- phosphate to glucose. Pyruvate Dehydrogenase Complex  Inside the mitochondrion, pyruvate is converted to acetyl-CoA by the pyruvate dehydrogenase complex. The net reaction is: pyruvate + NAD+ + CoA → Acetyl-CoA + NADH + H+ + CO2  This step is irreversible.  It involves the oxidative decarboxylation of pyruvate  The PDH complex consists of three catalytic enzymes: E1: Pyruvate dehydrogenase E2: An acetyltransferase (dihydrolipoamide acetyltransferase) E3: Another dehydrogenase (dihydrolipoamide dehydrogenase) These enzymes are arranged exquisitely in the multienzyme complex such that the substrates for one enzyme is passed directly to the next enzyme in the sequence, in a series of well controlled and efficient steps. Pyruvate Dehydrogenase Complex  The E1 component of the PDH complex is thiamine (Viatmin B1)-dependent.  The E2 component has bound lipoamide, a derivative of the vitamin, lipoic acid.  Coenzyme A, a sulfur-containing molecule, is derived from another vitamin, pantothenic acid (pantothenate).  The E3 is a flavoprotein (contains FAD). FAD is derived from Vitamin B2 (riboflavin).  The PDH complex is even more sophisticated because it contains other proteins and enzymes, including:  A structural protein, E3-binding protein (E3BP)  Two regulatory protein kinases (pyruvate dehydrogenase kinase) which phosphorylate the pyruvate dehydrogenase (E1) at serine residues. Phosphorylation decreases activity of the E1 dehydrogenase.  A regulatory protein phosphatase, pyruvate dehydrogenase phosphatase, which removes the phosphates added by the kinases. Dephosphorylation increases activity of the E1 dehydrogenase.  It is even more complex than this, because there are 4 different isoforms of the protein kinases and 2 isoforms of the protein phosphatase. Pyruvate Dehydrogenase Complex  In the first step (catalyzed by E1), pyruvate is first decarboxylated to release carbon dioxide, then there is reduction and acetylation of the lipoamide component of E2.  In the second step, the acetyl moiety is transferred from E2 to Coenzyme A ( a substrate of the reaction), to form acetyl-CoA.  At this stage, the acetyl-CoA has been produced as well as carbon dioxide, but the lipoamide prosthetic group is in a reduced form.  To regenerate the lipoamide in E2 to its original state, it transfers electrons to the E3 component. E3 contains FAD. Thus reduces the FAD to FADH2.  To return E3 to its original state, the FAD must be regenerated. This is achieved by E3, which transfers hydrogens from FAD to NAD+ to produce NADH.  The NADH can then enter the electron transfer chain to be used for ATP synthesis by oxidative phosphorylation.  decarboxylase FADH2 + NAD+ -> FAD + NADH + H+ dehydrogenase Acetyl transferase Pyruvate Dehydrogenase Multienzyme Complex Pyruvate Dehydrogenase Complex  The acetyl-CoA produced from the pyruvate can now enter the Krebs cycle.  Further energy has been “tapped” from the pyruvate molecule as reducing equivalents in the form of NADH, and these reducing equivalents can, in turn, be “tapped” as electrochemical energy, which can be “tapped” eventually as phosphodiester bond energy of ATP.  The structure/composition of the pyruvate dehydrogenase complex varies from tissue to tissue due to different modifications (e.g. phosphorylation) of its components and different isoforms of its proteins. This allows integration of metabolism between different tissues and exquisite control of central metabolism according to the needs of specific tissue and needs of the body as a whole.  What happens in thiamine (Vitamin B1) deficiency to the intermediates of glycolysis? ATP yield for oxidative metabolism of glucose ATP yield for oxidative metabolism of glucose Krebs Cycle Steps 1 and 2: Citrate synthase converts oxaloacetate and acetyl-CoA (which contains the atoms and bonds from the original glucose molecule) to citrate and aconitase converts citrate to isocitrate. CoA citrate synthase H20 oxaloacetate aconitase citrate isocitrate Steps 3 and 4: Isocitrate dehydrogenase converts isocitrate to alpha-ketoglutarate, converting NAD + to NADH. Alpha-ketoglutarate dehydrogenase complex then converts alpha-ketoglutarate to succinyl-CoA, using Coenzyme A and NAD+ as substrates, generating carbon dioxide and NADH. isocitrate NAD+ Isocitrate NADH dehydrogenase CO2 CoA alpha-ketoglutarate alpha-ketoglutarate dehydrogenase CO2 NAD+ NADH succinyl-CoA Steps 5 and 6: Succinyl-CoA synthetase (also called succinyl thiokinase) converts succinyl-CoA to succinate: this is a substrate-level phosphorylation that generates GTP, which is converted to ATP. Succinate dehydrogenase, a component of Complex II of the electron transport chain, containing FAD, fumarate Succinyl-CoA synthetase Succinate CoA dehydrogenase FADH2 FAD succinate Pi GTP GDP Succinyl-CoA ADP ATP Steps 7 and 8: Fumarase converts fumarate to malate. Then malate dehydrogenase converts malate to oxaloacetate, generating NADH, and providing oxaloacetate for another cycle of the Krebs cycle. NADH Oxaloacetate combines malate with acetyl-CoA to form dehydrogenase citrate, and begin another NAD round of the Krebs cycle. oxaloacetate malate fumarase H2 0 fumarate The Krebs Cycle  The Krebs cycle (citric acid cycle; tricarboxylic acid cycle) consists of 8 enzyme-catalyzed steps.  Most of the energy from the glucose molecule still has to be “tapped” by the time glucose has been converted to acetyl-CoA, the product of the pyruvate dehydrogenase complex in mitochondria.  Most of the energy from glucose (or other food fuels, including fats) is “tapped” by the Krebs cycle as NADH or FADH2 reducing equivalents: these can then be used to generate ATP from the electron transport chain/ oxidative phosphorylation.  There is one substrate-level phosphorylation, at the succinyl-CoA synthetase (succinyl thikinase) step. This step generates guanosine triphosphate (GTP) from GDP. The GTP formed can be converted to ATP by the enzyme, nucleotide diphosphate kinase: GTP + ADP  ATP + GDP The Krebs Cycle: Where is the energy?  Steps in the Krebs cycle that “tap” energy from the glucose molecule:  NADH is produced at 3 steps:  Isocitrate dehydrogenase  Alpha-ketoglutarate dehydrogenase  Malate dehydrogenase  FADH2 is produced at one step:  Succinate dehydrogenase  Substrate-level phosphorylation forms ATP (via GTP) at one step:  Succinyl-CoA synthetase  Electron transport chain and oxidative phosphorylation  When glucose has been metabolized through glycolysis/ pyruvate dehydrogenase complex/ Krebs cycle, the only “real” ATP has come from substrate-level phosphorylation (2 steps in glycolysis and 1 step in Krebs cycle).  Most of the energy tapped from the glucose molecule is in the form of reducing equivalents, as NADH and FADH2. This “redox” energy has yet to be converted to the chemical energy of ATP.  The reducing equivalents from glycolysis, pyruvate dehydrogenase complex and Krebs cycle are directed to the electron transport chain, which converts the reducing energy to a proton gradient across the inner mitochondrial membrane. This creates a proton motive force across the membrane. Peter Mitchell proposed this idea in 1961 and it has been proven to be correct. It is known and the Chemiosmotic Theory.  The protons are pumped by the components of the electron transport chain from the mitochondrial matrix to the space between the inner and outer mitochondrial membranes.  Electron transport chain and oxidative phosphorylation  The electron transport chain components are embedded in the inner mitochondrial membrane, though ubiquinone (Coenzyme Q) and cytochrome c is highly mobile and cytochrome c is a soluble protein.  There are four large multi-subunit complexes, Complexes I, II, III and IV. The ATP synthase (also known as the F0F1-ATPase, which converts ADP and inorganic phosphate to ATP, is also known as Complex V. Complex I: (NADH-Q oxidoreductase) Complex II: (Q-cytochrome c oxidoreductase) Complex III: (succinate- Q reductase) Complex IV: (cytochrome c oxidase)  Proton pumping across the membrane occurs with Complex I, III and IV.  Flavoproteins (FMN/FAD); iron-Sulphur proteins; heme proteins; coenzyme Q; are involved in transfer of electrons down a gradient, terminating in reduction of oxygen to form water.  Electron transport chain and oxidative phosphorylation  More protons are pumped form NADH than for FADH2 oxidation, so more ATPs are made from NADH.  Uncouplers such as dinitrophenol and UCP1 (uncoupling protein 1) “uncouple” electron transport from ATP formation by dissipating the proton gradient.  The electron transport chain (especially Complex I and III) is the main source of reactive oxygen species (ROS) in a cell and these, if excessive, can cause damage to cellular components. Electron transport chain and oxidative phosphorylation Electron transport chain and oxidative phosphorylation Electron transport chain and oxidative phosphorylation  Gluconeogenesis, Pentose Phosphate Pathway and glycogen metabolism bbBy :Elias T/mariam (BSc,MSc) 1  Gluconeogenesis  Gluconeogenesis is the generation of glucose from non-carbohydrate substrates.  Main substrates for gluconeogenesis are pyruvate, lactate, glycerol, alanine and glutamine and other gluconeogenic amino acids.  Gluconeogenesis occurs when glucose is in relative short supply in the diet and so needs to be made by the body to maintain blood glucose levels and supply of glucose for glucose-requiring tissue, such as red blood cells, kidney medulla, testes and the brain, which needs glucose but can also use ketone bodies.  In humans, gluconeogenesis occurs mainly in the liver and to some extent in the kidney cortex and small intestines. These cells contain glucose-6-phosphatase, which allows glucose-6- phosphate to be converted to glucose and released into the blood stream. Muscle lacks glucose- 6-phosphatase, for example, so cannot supply glucose to the bloodstream.  Gluconeogenesis requires ATP and GTP to drive it: it is endergonic 2  Gluconeogenesis  Gluconeogenesis is not a direct reversal of glycolysis because three steps in glycolysis are irreversible:  Hexokinase (glucose to glucose-6-phosphate)  Phosphofructokinase (fructose-6-phosphate to fructose-1,6-bisphosphate)  Pyruvate kinase (phosphoenolpyruvate to pyruvate)  Therefore, “new” enzymes are needed to reverse gluconeogenesis. There are 4 enzymes that are unique to gluconeogenesis that bypass the 3 irreversible enzymes of glycolysis:  Pyruvate carboxylase (pyruvate to oxaloacetate)  Phosphoenolpyruvate carboxykinase (PEPCK) (oxaloacetate to phosphoenolpyruvate)  Fructose-1,6-bisphosphatase (fructose-1,6-bisphosphate to fructose-6-phosphate)  Glucose-6-phosphatase (glucose-6-phosphate to glucose)  The remaining enzyme steps are reversible and so are the same in both glycolysis and gluconeogenesis. 3 Gluconeogenesis Enzymes that are unique to glycolysis (left) are shown in red. Enzymes unique to gluconeogenesis (right) are shown in blue. 4 Gluconeogenesis: Pyruvate carboxylase and PEPCK 5 Gluconeogenesis: Fructose-1,6-bisphosphatase and glucose-6-phosphatase 6  Glucose cannot be made from fatty acids in humans by gluconeogenesis!  Whereas glucose can be converted to fatty acids (via glycolysis, pyruvate dehydrogenase complex and acetyl-CoA, the precursor of fatty acid synthesis), acetyl- CoA and fatty acids cannot be converted to glucose.  In starvation, therefore, fatty acids (from stored fats) can be used as fuel by many tissues, but not neurons in the brain, which can use only glucose or ketone bodies. Ketone bodies are produced in the liver from fatty acids. 7 Gluconeogenesis from lactate  Lactate can be converted to glucose by gluconeogenesis by first being converted to pyruvate (lactate dehydrogenase).  The pyruvate then enters mitochondria, where it is converted to oxaloacetate by pyruvate kinase. Pyruvate kinase cannot function without acetyl-CoA, which is an obligate activator of the enzyme.  In the mitochondrion, oxaloacetate is converted to malate, which enters the cytosol and is converted to oxaloacetate in the cytosol. The oxaloacetate is then converted to phosphoenolpyruvate (PEP) by PEPCK.  The PEP can now be converted to glucose by the rest of the gluconeogenesis pathway.  Lactate dehydrogenase reaction supplies the NADH for the glyceraldehyde-3-phosphate dehydrogenase step to form glyceraldehyde-3-phosphate.  Lactate from red blood cell glycolysis can be made into glucose, mainly in the liver, by gluconeogenesis.  Lactate production in muscle can be cleared and resynthesized into glucose by liver 8 gluconeogenesis, and the glucose can be reused by skeletal muscle as a source of energy. 9 Gluconeogenesis and the Cori cycle: When there is excessive build-up of lactate in skeletal muscle, the lactate enters the bloodstream and is taken up by the liver, where it undergoes gluconeogenesis: the glucose so formed can then be reused by the muscle. This is called the Cori cycle. 10 Glucose-Alanine cycle: Pyruvate can be converted to alanine by transamination in muscle and other tissues. The alanine enters the bloodstream and is taken up by the liver, where its undergo gluconeogenesis to glucose and its amino group gets converted to urea by the urea cycle for excretion. The glucose formed from the alanine can enter the bloodstream and be used as a fuel by other tissues. The glucose-alanine cycle therefore serves to allow excretion of nitrogen from amino acids as well as to produce glucose from the carbon skeleton of alanine. 11 12 Glucogenic and ketogenic amino acids. Amino acids are catabolized to form pyruvate or Krebs cycle intermediates. Some amino acids are glucogenic and their metabolic products can be used to make glucose by gluconeogenesis (purple). Some amino acids are ketogenic and are metabolized to acetyl-CoA or acetoacetyl-CoA, and can be used to make ketone bodies or undergo metabolism by the Krebs cycle (light blue). Some amino acids are both glucogenic and ketogenic because they form intermediates that can be metabolized in both ways. Two amino acids (leucine and lysine) are ketogenic only (not glucogenic). 13 Gluconeogenic and ketogenic amino acids  Amino acids that are ketogenic only (not gluconeogenic):  Leucine  Lysine  Amino acids that are both gluconeogenic AND ketogenic (5 of them):  Phenylalanine  Tyrosine  Tryptophan  Isoleucine  Threonine  Amino acids that are gluconeogenic only (13 of them):  Glycine Serine Valine Histidine Arginine  Cysteine Proline Alanine Aspartate Glutamate  Asparagine Glutamine 14 Methionine Pentose Phosphate Pathway (Hexose Monophosphate Shunt, or Phosphogluconate Pathway)  The pentose phosphate pathway occurs in the cytosol.  It has two major functions:  1) To make NADPH  2) To make the pentose, ribose-5-phosphate, which is a component of RNA and is the source also of deoxyribose for DNA.  NADPH is involved in many biosynthetic pathways, for example, steroid, fatty acid and catecholamine synthesis.  NADP is also involved in protecting cells from damage by reactive oxygen species (ROS).  No ATP is produced (unlike glycolysis).  NADPH is produced (not NADH, as in glycolysis)  Carbon dioxide is produced (not in glycolysis). 15 Pentose Phosphate Pathway (Hexose Monophosphate Shunt, or Phosphogluconate Pathway)  The starting molecule for the pentose phosphate pathway is glucose-6-phosphate, an intermediate in glycolysis.  There are two phases:  An oxidative, irreversible phase, in which two NADPH molecules and one carbon dioxide molecule are generated. The product of this phase is ribulose-5-phosphate.  A non-oxidative phase, in which glucose-6-phosphate is resynthesized from ribulose-5-phosphate  The first step is catalyzed by glucose-6-phosphate dehydrogenase (G6PDH). This step and the 6- phosphogluconate dehydrogenase step, produce NADPH from NADP+.  For every 6 molecules of glucose-6-phosphate that enter the pathway, five glucose-6-phosphate molecules are produced (hence it is a cycle) and six CO2 are produced. One carbon is lost (as carbon dioxide) for each cycle. 16 Pentose Phosphate Pathway (Hexose Monophosphate Shunt, or Phosphogluconate Pathway)  In the non-oxidative phase, ribulose-5-phosphate is converted to glucose-6-phosphate.  This phase interconverts various sugars reversibly using two enzymes, transketolases and transaldolases.  Important pentose phosphates (especially ribose-5-phosphate and xylulose-5-phosphate) are produced in this second phase.  One way to think of the pentose phosphate pathway is to consider it as a cycle with two phases. In the first phase, glucose-6-phosphate is oxidized in two steps to produce ribulose-5-phosphate and two NADPH molecules plus carbon dioxide. The key in this step is formation of NADPH, which can be used for biosynthetic pathways and for its roles in free radical protection. The second phase involves regeneration of glucose-6-phosphate, but especially it makes ribose-5- phosphate for nucleotide and nucleic acid synthesis. It also allows various sugars (4,5,6 and 7=carbon sugars) to be interconverted to each other. 17  CO2 NADPH NADPH Ribose-5-P Ribose-5-P 18 Pentose Phosphate Pathway: Summary of Steps 19 Glycogen metabolism Glycogen structure: Alpha-D-glucose residues joined by alpha(1->4) linkages with branches joined to the molecule as alpha(1->6) linkages. Glycogen Metabolism  Glycogen is stored as large particles in liver and muscle cells. The glycogen molecules are highly branched and contain 10,000 to 50,000 glucose residues in size. The glycogen particles contain all of the enyzmes and proteins involved in synthesis and degradation of glycogen, plus the regulatory enzymes and proteins of glycogen metabolism.  Glycogenesis: synthesis of glycogen.  Glycogenolysis: breakdown of glycogen.  Glycogenesis and glycogenolysis are coordinately controlled and also reciprocally regulated such that when one goes up the other goes down to maintain the body’s glycogen needs.  While glycogen is the storage form of glucose (carbohydrate) in humans, fat is the main storage form of fuel. Glycogen is used up rapidly during starvation (within a day), whereas fat can continue to supply energy for weeks, even months. Glucose is always required at some level by the body, and is made by gluconeogenesis when dietary glucose is lacking.  Why is fat a better storage form of energy than carbohydrate? Fat produces more energy per gram following catabolism than carbohydrate. (9 kcal/g for fat compared with 4 kcal/g for carbohydrate). Fat (being hydrophobic) is stored in an “anhydrous form,” whereas glycogen is stored as a hydrated from consisting of 2/3 water content. This means that for every 3 g of (hydrated) glycogen stored, 2 g of it consists of water and only one gram is molecular glycogen. Therefore, for every g of glycogen stored there is about 4 kcal of energy, whereas for every g of fat stored there is about 27 g of energy. If food energy was stored as glycogen rather than fat, survival of starvation would be much shorter, OR body weight would have to be extremely high to compensate- we would be too heavy function. Glycogenesis (glycogen synthesis)  Glycogen is synthesized initially by phosphorylation of glucose to glucose-6-phosphate. In the liver, the enzyme is glucokinase. In muscle, the enzyme is hexokinase. Both enzymes achieve the same chemical end-point. Glucose + ATP Glucose-6-phosphate + ADP  In the second step, glucose-6-phosphate is converted to glucose-1-phosphate by phosphoglucomutase. Glucose-6-phosphate Glucose-1-phosphate Phosphoglucomutase is the only enzyme common to glycogenesis and glycogenolysis. Uridine diphosphate glucose (UDP-glucose) is formed from glucose-1-phosphate during glycogenesis. Formation of UDP-glucose: The enzyme, UDP-glucose pyrophosphorylase catalyses the reaction of glucose-1-phosphate with UTP to form UDP-glucose. Note that pyrophosphate (PPi) formed is rapidly converted to phosphate by pyrophosphatase and this drives the reaction in one direction. UDP-glucose serves as the molecule that “adds” a glucose moiety to the growing glycogen molecule during synthesis. A primer is needed for this process: this primer is made on a protein called glycogenin, which is part of the glycogen molecule. Glycogenin is glucosylated by UDP glucose on a tyrosine residue hydroxyl group of glycogenin. Glycogenin is itself an enzyme that phosphorylates itself by autocatalysis. Further glucose residues are added (by glycogenin autocatalysis) to form a chain of 6 or 7 glucose units joined by alpha(1->4) links. Glycogen synthesis (glycogenesis)  After about 6 or 7 glucose residues, to form a glucan chain attached to glycogenin, have been added to glycogenin, glycogen synthase takes over the process.  Glycogen synthase then adds further glucose residues as alpha(1->4) units, using UDP-glucose as a substrate.  Branching enzyme creates the alpha(1->6) branches of glycogen by transferring groups of at least 6 residues from one chain to a nearby residue or a residue in the same chain, creating branch points.  Each branch point then grows as more glucose residues are added from UDP-glucose in alpha(1- >4) linkages. At the same time, branching enzyme creates branches at new branch points. This continues till molecules up to 50,000 or more glucose residues are made. Glycogen synthesis (glycogenesis): Growing chains with branch points and branches Glycogen synthesis (glycogenesis): further elongation and branching occur with glycogen synthase and branching enzyme to create a large glycogen molecule. Glycogenolysis (glycogen breakdown)  At least 3 enzymes are involved in breakdown of glycogen into monosaccharide (glucose-1- phosphate) subunits.  Glycogen phosphorylase (the rate-limiting step) hydrolyses alpha(1->4) bonds between glucose molecules to form glucose-1-phosphate. This requires pyridoxal phosphate as a coenzyme.  Glycogen phosphorylase removes residues from the terminal branches, sequentially until there are 4 glucose residues joined to an alpha(1->6) branch point.  Then the enzyme, glucan transferase, transfers trisaccharide (3 glucose residues) at a time from the branch point to another one. This exposes the branch point, which is recognized by another enzyme, alpha-1,6-glucosidase (debranching enzyme), which removes the one residue joined to the branch point with an alpha(1->6) link, releasing glucose as a product Glycogenolysis: Glycogen phosphorylase removes a terminal glucose residue, forming glucose-1-phosphate Glycogenolysis: Glucose-1-phosphate released from glycogen by glycogen phosphorylase is converted to glucose-6-phosphate by phosphoglucomutase. Glucose-6-phosphatase in liver can convert the glucose-6-phosphate to glucose. Glucan transferase then transfers 3 glucose residues as a trisaccharide to the end of another branch, leaving a single glucose residue joined by alpha(1->6) linkage. This residue is removed by debranching enzyme (alpha-1,6-glucosidase) to produce glucose. Glucan transferase Debranching enzyme METABOLIC HOMEOSTASIS LIPID METABOLISM BY: G/hiwot G. Asst.prof. of Medical Biochemistry Aksum University College of Health Sciences 1  Module out line Introduction to Bioenergetics Biochemical Catalysis and Enzymes (2 hrs.) Mechanisms of enzyme catalysis, enzyme kinetics with emphasis on the importance of Vmax and Km, the effects of pH and temperature; enzyme inhibition (2hrs) Enzyme regulation and the clinical application of enzymes, Coenzymes, cofactors and their functions (2hrs) Enzymes and transporters involved in the digestion and absorption of carbohydrates (2hr) Glycolysis and its clinical significance in different cells (2hr) Hexose monophosphate pathway and glucuronic acid pathway, and their clinical significance in different cells (2hrs) Regulation of the pentose phosphate pathway; the glucuronic acid pathway and its functions; Metabolism and clinical significance of other hexose sugars (2hr) 2  Module out line cont… Glycogen storage sites, synthesis and degradation; regulation of glycogen metabolism (1 hr.) The significance of gluconeogenesis and responsible organs, reactions and regulation and the Cori cycle. The Pyruvate Dehydrogenase Reaction (1 hr.) Electron Transport and Oxidative Phosphorylation: The link between redox potential and free energy, components of the respiratory chain, coupling of electron transport and oxidative phosphorylation, the energy yield of reduced coenzymes, inhibitors and uncouplers (2 hr.) 3  Module out line cont… Metabolism of micronutrients (4hours) Nucleotide Metabolism (4hrs.): Purine metabolism: de novo and salvage pathways, degradation; Pyrimidine metabolism; Inborn errors of nucleotide metabolism Dietary lipid utilization by cells and fatty acid oxidation (2hr) Fatty acid biosynthesis (1hr) Lipogenesis, and lipolysis with their clinical importance (1hr) Ketone body production and utilization (1hr) The synthesis and degradation of phospholipids, sphingolipids and glycolipids, and their clinical significance (1.5 hr.) Lipoprotein metabolism and related disorders (1.5 hr.) 4  Module out line cont… Proteolytic enzymes, protein turnover, nitrogen balance: lysosomal and the ubiquitin/proteasome pathway Amino acid metabolism (catabolism) and their clinical significance TCA Cycle and its regulations Amino Acid Derived Nitrogenous Compounds metabolism and their clinical significance; Inborn Errors of Amino Acid Metabolism (2hr) Ammonia metabolism (The urea cycle) and its clinical significance and related metabolic inborn errors (2hr). The packaging of DNA in chromosomes, the nucleoid; Prokaryotic and eukaryotic DNA replication Mutations and DNA repair mechanisms; Prokaryotic and eukaryotic gene transcription, Translation Recombinant DNA and cloning, gene therapy, DNA amplification, DNA fingerprinting 5  Learning Objectives Once the lecture is finished, You should be able to  Describe the digestion, absorption and transportation of lipids  Explain the metabolic processes and regulation of fatty acid and triglyceride synthesis beta oxidation 6 7  Lipids are a heterogeneous group of water- insoluble (hydrophobic) Organic molecules. Hydrophobicity is due to the long – hydrocarbon chains.  Common classes of lipids include: 1. Fatty acids. 2. Triacylglycerols. ( 3 FAs esterified to glycerol). 3. Phospholipids. (phosphoglycerides), (the alcohol moeity glycerol + phosphate +2 FAs + other groups) 4. Steroids. (cholesterol, steroid hormones, bile acids, vitamin D) 5. Glycospingolipids. (the alcohol moiety is sphingosine + fatty acid + carbohydrate). 8 Structures of the common classes of lipids, the hydrophobic parts of 9 the molecules are in an orange colour  Functions of lipids i. Efficient energy sources. ii. To store energy in the body iii. thermal insulator against low temperatures. iv. Components of biological membranes and nerve tissues: e.g., phospholipids, glycolipids and sterols v. Lipids in myelinated nerves acts as insulators for propagation of depolarization wave (action potential). vi. Precursor for hormones. vii. Emulsifying agents in digestive tract 10 viii. Fats are essential for the absorption of fat-soluble vitamins ix. Component of lung surfactant (used to reduce surface tension) Dipalmitoyllecithin x. Precursor for intracellular messengers like PIP3 & DAG xi. Play crucial roles as enzyme cofactors and electron carriers: e.g., ubiquinone  Knowledge of lipid biochemistry is necessary in understanding many important biomedical areas, eg, obesity, diabetes, atherosclerosis, fatty liver, and the role of various polyunsaturated fatty acids in nutrition and health. 11  Digestion and absorption of dietary Lipids  Adult daily intake ~ 60-150 grams The main lipids in diets are triacylglycerols, which accounts for about 90% dietary lipids, because they are the major storage lipids in the plants and animals. In addition to TAGs, phospholipids, glycolipids, cholesterol, cholesterylesters, free fatty acids, carotenes, and other fat-soluble vitamins are present in the diet we eat.  Sources of dietary lipids include: Plants: cooking vegetable oils Animals: Butter, animal fats, milk, cheese, egg yolk, meat. 12  Digestion of lipids is hydrolysis of TAG, complex lipids, and cholesterylesters into fatty acid, 2- monoacylglycerols, and free cholesterol.  Lingual lipase, gastric lipase, pancreatic lipase and intestinal lipase are involved in digestion of TAGs – the most active and important being the pancreatic lipase.  Digestion of lipids begins in the stomach by acid stable lingual lipase (produced by cells at the back the tongue: Ebner’s gland) and gastric lipase (secreted by gastric mucosa).  Lingual and gastric lipases have an acidic pH optimum which ranges from 3-6. 13  Lingual and gastric lipase preferentially hydrolyze TAGs containing short- and medium-chain (12 or fewer carbon atoms) fatty acids to form free fatty acids and diacylglycerols.  These enzymes play important role in lipid digestion in infants, who take relatively large amounts of milk. Milk contains TAGs with high percentage of short- and medium- chain fatty acids.  Lingual and gastric lipases are also important in individuals with pancreatic insufficiency (e.g., cystic fibrosis).  Overall in adults, dietary lipids are not digested to any extent in the mouth or the stomach but rather progress more or less intact to the small intestine. 14  Digestion in the small intestine:  Small intestine is the major site of lipid digestion using pancreatic enzymes  This begins in the duodenum, when the entrance of the acid chyme from the stomach stimulates the secretion of enteric hormones by the duodenal mucosa.  The low pH (HCl) of the chyme entering the intestine stimulates the secretion of secretin, which stimulates the pancreas to release bicarbonate rich juice into the duodenum.  The bicarbonate helps neutralize the pH of the intestinal contents, bringing them to the appropriate pH for the digestive activity of pancreatic enzymes 15  Secretin also has inhibiting effect on the gastric secretion. The decrease in gastric secretion is important to slow the passage of chyme from stomach when the small intestine is already filled.  Cells in the mucosa of the jejunum and lower duodenum produce cholecystokinin (CCK, also known as pancreozymin), in response to the presence of lipids and partially digested proteins entering the intestine.  CCK acts on the exocrine cells of pancreas (pancreatic acini) and cause secretion of pancreatic digestive enzymes.  CCK acts on the gallbladder causing it to contract and release bile.  In addition, CCK decreases gastric motility, resulting in a slower release of gastric contents into the small intestine. 16 17  Emulsification of lipids in the small intestine  Emulsification is breakdown of large fat globules into smaller ones to increase the available surface area of lipids for enzyme action.  Most of the emulsification of dietary lipids occurs in the duodenum under the influence of bile, which contains bile salts and phospholipids.  These emulsifying agents interact with both the lipid particles and the aqueous duodenal contents, thereby stabilizing the particles as they become smaller and preventing them from coalesing.!!  This increases the surface area of lipid droplets so that digestive enzymes work effectively at the interface of the droplet and the surrounding aqueous medium. 18  Emulsification is done by two complementary mechanisms:  Chemical dispersion of the lipid globules by detergent (amphipathic) properties of bile salts which are synthesized in the liver and stored in the gall bladder  Mechanical churning of food due to peristalsis  Bile acids are derivatives of cholesterol that have hydroxyl groups on the steroid nucleus and a shortened hydrocarbon chain with a carboxyl end.  The term bile salt refers to conjugated bile acids, which contain either glycine or taurine linked via amide to the carboxyl group of bile acid. 19   The bile salts are more amphipathic than bile acids and thus more effective emulsifiers. Bile Acids O C O R2 R1 CH3 CH3 HO OH H R1 = OH or H R2 = H or NHCH2COOH or NHCH2CH2SO3 20  The role of bile salts in emulsions and micelles Figure 21-20  Digestion of dietary lipids by pancreatic enzymes a) Digestion of TAGs by pancreatic lipase  Pancreatic lipase is found in enormous quantity in pancreatic juice and is highly efficient catalytically.  Pancreatic lipase requires bile salts, phospholipids, colipase and calcium for its maximal action.  Any deficiency of one of these factors cause inadequate fat digestion. For example, in obstructive jaundice fat digestion is impaired since there is obstruction of bile flow and hence absence of bile salts for emulsification of lipids. 22  Colipase is a non-enzyme protein produced by the pancreas as procolipase, which is activated in the intestine by trypsin.  Pancreatic lipase has low affinity towards emulsion particles of dietary lipids and bile salts. Colipase binds the pancreatic lipase in 1:1 ratio, and anchors the lipase at the lipid aqueous interface, thereby increasing the lipase activity.  Pancreatic lipase preferentially hydrolyses the fatty acids at C-1 and C-3, producing two free fatty acids and 2-monoacylglycerol 23  The enterocytes of the small intestine also produce a lipase known as enteric lipase that digest TAGs, but less important. 24  b) Digestion of cholesterylester  Most dietary cholesterol is present in free form, which undergo no digestion and absorbed as such.  10-15% of dietary cholesterol is esterified with long- chain fatty acids  Pancreatic juice contains cholesterol esterase which hydrolyzes the ester bond between cholesterol and fatty acid, producing cholesterol and free fatty acid. 25  c) Digestion of Phospholipds  Pancreatic juice also contains enzymes that act on phospholipids: phospholipase A2 and lysophospholipase  Phospholipase A2 is produced as proenzyme prophospholipase A2 and like procolipase is activated in intestinal lumen by trypsin.  Phospholipase A2 removes fatty acid at the C-2 position of a phospholipid, producing lysophospholipid and free fatty acid. 26 27  Action of Phospholipases Phospholipase A1 Phospholipase A2 Phospholipase C Phospholipase D  Although it is specific to fatty acids at the C-2 position of phospholipids, phospholipase A2 can digest phospholipids with d/t head groups and d/t FA chain length.  Phospholipase A2 requires bile salts (like cholesterol esterase) and Ca2+ ion for its optimum activity  Note that pancreatic phospholipase A2 acts on both dietary phospholipids and phospholipids secreted by liver and contained in the bile.  Lysophospholipase acts on lysophospholipids to remove the fatty acid at C-1, producing free fatty acid and glycerylphosphoryl base (e.g., glycerylphosphoryl choline) that may be excreted in feces, further degraded or absorbed. 29  Physiologically important lipases Lipase Site of action Preferred substrate Product(s) Mouth , Lingual / acid stable lipase stomach TAGS with medium chain FAS FFA+DAG Pancreatic lipase + co-lipase Small intestine TAGS with long chain FAS FFA+2MAG Intestinal lipase with bile acids Small intestine TAGS with medium chain FAS 2FFA+glycerol Unsat FFA Phospholipase A2 + bile acids Small intestine PLs with unsat. FA at position 2 lysolecithin Lipoprotein lipase insulin (+) Capillary walls TAGs in chylomicron or VLDL FFA+ glycerol Hormone sensitive lipase Adipose cell TAG stored in adipose cells FFA+ glycerol 31  Absorption of dietary lipids (By micelles formation)  The products of lipid digestion include monoacylglycerol, lysophospholipids, free fatty acids, cholesterol, phosphate, and fat-soluble vitamins. These products of digestion are absorbed in the duodenum and jejunum.  The products of lipid digestion combine with bile salts and form mixed micelles - disk-shaped clusters of amphipathic lipids having only 3-6 nanometers in diameter.  The mixed micelles are soluble in the aqueous environment of the intestinal lumen and carry the products of lipid digestion to the brush border of mucosal cells (enterocytes) where they are absorbed into the intestinal epithelium.  Under normal conditions over 95% of dietary lipids is absorbed. 32  Short and medium chain-length fatty acids do not require the assistance of a micelle for absorption by the intestinal mucosa.  At the brush border of enterocytes, lipid digestion products, particularly free fatty acids and monoacylglycerols, diffuse out of the mixed micelles into the enterocytes, which is possible because the lipids are also soluble in the epithelium cell membranes. 33  Except the bile salts, the rest are absorbed by the duodenal and jejunal epithelial cells  The bile salts are left behind in the lumen of the intestine, where they function again and again for absorption of more lipids. 34  The bile acids are actively reabsorbed in the ileum and recirculated through the enterohepatic circulation to the liver. Enterohepatic circulation of bile salts 35  Resynthesis of TAGs, CE and PLs  Once within the enterocytes, the mixture of lipids absorbed migrates to the cell’s smooth endoplasmic reticulum, where they are used for re-synthesis of triacylglycerols, cholesterylesters and phospholipids.  The cytoplasmic fatty acid-binding proteins in the enterocytes, which have high affinity for long-chain fatty acids, transport fatty acids to the smooth endoplasmic reticulum for re-synthesis of TAGs, PLs and CEs. 36  Absorbed cholesterol is also reesterified to a fatty acid in enterocytes into cholesterylester by acyl CoA: cholesterol acyltransferase. ester 37  Not all of the absorbed cholesterol is esterified to form CE, but that in excess of which can be accommodated on the surface of chylomicron particles is esterified.  Note also that virtually all long-chain fatty acids entering the enterocytes are used in the synthesis of TAGs, cholesterylesters and phospholipids.  Within the enterocytes golgi appatratus, the resynthesized triacylglycerols, phospholipids, and cholesterylesters combines with apoproteins to form lipid-carrying particles called chylomicrons. 38  Chylomicrons are released by exocytosis from enterocytes into the lacteals (lymphatic vessels originating in the villi of the small intestine) through fenestrations.  The presence of chylomicrons in the lymph after lipid- rich meal gives the lymph a milky appearance, termed as chyle.  Through lymphatic vessels chylomicrons reach thoracic duct, which empties into the left subclavian vein, where they enter the bloodstream.  The major apoprotein associated with chylomicrons as they leave the small intestine is apolipoproteins B-48 (apo B-48). 39  Assembly and secretion of chylomicron by intestinal mucosal cells. [Note: Short- and medium-chain length fatty acids do not require incorporation into micelles and directly enter into the blood.] 1/17/2025 40  Chylomicrons are made of 89% triacylglycerols, 2% cholesterol, 8% phospholipids, and 1% protein. In addition, they contain fat-soluble vitamin (A, D, E and K)  The core of chylomicrons consists of triacylglycerols, cholesterylesters, and other absorbed lipophilic molecules such as fat-soluble vitamins. The surface of chylomicrons is a monolayer composed of phospholipids, free cholesterol, and apolipoproteins, primarily apo B-48 and apo C II  Short- and medium chain fatty acids and glycerol are water soluble, and do not require bile salts for their absorption. They are absorbed directly into the portal blood and transported to the liver bound with albumin. 42 Chylomicron str. 43 44 45 Use of dietary lipids by the tissues TAGs in chylomicrons are broken primarily in the skeletal muscles and adipose tissue by lipoprotein lipase (LPL) to glycerol and free fatty acids; and the chylomicrons shrink to become chylomicron remnants. Lipoprotein lipase Triacylglycerol + 3 RcooH  LPL is found on the luminal surface of endothelial cells of the capillary beds of the peripheral tissues. LPL is activated by apo C II LPL deficiency in familial lipoprotein lipase deficiency results in massive chylomicronemia. 46  Fate of free-fatty acids 1. Directly enter adjacent muscle cells or adipocytes. 2. Alternatively, free fatty acids may be transported in blood in association with serum albumin, until they are taken by cells. 3. Most cells can oxidize fatty acids to obtain energy (ATP). However, the brain and other nervous tissues, erythrocytes and the adrenal medulla cannot use plasma free- fatty acids for fuel, regardless of the blood levels of fatty acids. 4. Adipocytes can also re-esterify free-fatty acids to produce triacylglycerol molecules, which are stored until the fatty acids are needed. 47  Fate of glycerol Glycerol is used by the liver to produce glycerol-3- phosphate, which can enter glycolysis or gluconeogenesis by oxidation to dihydroxyacetone phosphate (DHAP).. 48  Fate of chylomicron remnants The remnants are taken up by the liver where, they are hydrolyzed to their component parts. This is due to the presence of apolipoprotein E on the surface of chylomicrons which has apolipoprotein E receptors in hepatic cells plasma membranes. Deficiency of apolipoprotein E, leads to familial type III hyperlipoproteinemia which leads to defective removal of chylomicron -remnants from the plasma 49 50  Abnormalities of lipid digestion and absorption Steatorrhea: is a condition in which there is an abnormal increase in stool lipid excretion (>6% of dietary lipid).  Normally more than 95% of ingested lipid is absorbed (less than 5% of dietary lipid is excreted in feces). Fat soluble vitamins are also excreted in feces.  Steatorrhea is caused by one or more defects in digestion and absorption of dietary lipid:  Defective digestion: due to deficiency of pancreatic lipase as a result of chronic pancreatitis, cystic fibrosis, severe gastric hyperacidity causing acidic duodenal pH (Zollinger- Ellison syndrome), obstruction of pancreatic duct by tumors and pancreatectomy.  Feacal fat is mostly undigested TAGs. 51  Defective absorption: due to deficiency of bile salts as a result of:  bile duct obstruction as in tumours or stones of the bile duct and in some cases of hepatitis and liver cirrhosis.  Feacal fat is in the form of 2-monoacylglycerol.  Also it may be due to defective intestinal mucosal cells as in celiac sprue (inflammation of the small intestinal mucosa) 52 53 Chyluria It is characterized by excretion of milky urine. It is due to abnormal connection between urinary tract and lymphatics of small intestine.  It is also called as chylous fistula.  It disappears when dietary fat is replaced with fat containing short chain and medium chain fatty acids. Congenital abeta lipoproteinemia It is of genetic origin. Triglycerides accumulates in intestinal cells due to lack of apoB-48 required for lipoprotein formation (chylomicrons). 54 Cholestasis Lipid digestion and absorption is impaired in intra or extra hepatic cholestasis due to non availability of adequate amounts of bile salts, phospholipids and cholesterol.  In cholestatic patients, liquid crystal vesicles are formed instead of mixed micelles. Proper biliary secretion of phospholipid is necessary for chylomicron formation in enterocyte and secretion of lipids into lymph. Essential fatty acid deficiency (EFAD) It occurs in cholestatic patients due to malabsorption of lipids.  EFAD during cholestasis itself can impair efficient lipid absorption and transport. Because proper biliary secretion of phospholipid is necessary for formation of mixed micelles and chylomicrons. 55 56  De-novo synthesis of fatty acids substrate: acetyl-CoA, NADPH + H+ product: palmitate (= endproduct of FA synthesis) function: de novo synthesis of FA which are stored as TAG subcelullar location: cytosol organ location: mainly liver and adipose tissue and also other tissues regulatory enzyme: acetyl-CoA carboxylase (ACC) 1/17/2025 57  De-novo synthesis of fatty acids  Fatty acids are used for energy source, synthesis of membrane and other important lipids  Fatty acids are the preferred source of energy for the heart, the liver and for the skeletal muscles (during rest)  Fatty acids Can be supplied from:  the diet.  Synthesis from acetyl-CoA molecules (de novo) derived from excess dietary carbohydrates and amino acids in a well- fed state  Degradation of stored TAG (lipolysis)  In humans, de-novo FA synthesis occurs primarily in the liver, lactating mammary gland, and adipocytes. 58  De novo FA synthesis is a cytosolic process whereas most of the starting molecule (acetyl CoA ) is obtained from mitochondrial reactions.  The sources of mitochondrial acetyl CoA are the oxidation of pyruvate, degradation of fatty acids, ketone bodies or amino acids.  The Coenzyme A portion of acetyl CoA cannot cross the mitochondrial membrane, only the acetyl portion is transported to the cytosol in the form of citrate.  Citrate lyase cleaves citrate giving cytosolic acetyl- CoA and oxaloacetate  Protein degradation (transamination) provides mainly cytosolic acetyl CoA 59 FA synthesis 60  Fatty acid synthesis incorporates carbons from acetyl CoA into a growing fatty acid chain, utilizing ATP and NADPH.  The NADPH is provided mainly from PPP and from malic enzyme as well.  The first step is the rate limiting step of fatty acid synthesis which involves the synthesis of malonyl- CoA by carboxylation of acetyl-CoA using acetyl- CoA carboxylase (ACC) and biotin coenzyme. 61 Structure of ACC 62  A. Short term regulation of ACC includes: a. Allosteric regulation of acetyl CoA carboxylase:  Citrate causes polymerization of the enzyme leading to its activation, while malonyl CoA and long chain fatty acyl CoA such as palmitoyl CoA (the end product of the pathway) depolymerize the enzyme leading to its inactivation. b. Reversible phosphorylation:  In the presence of epinephrine, the enzyme is phosphorylated and thereby inactivated and in the presence of insulin, the enzyme is de-phosphorylated and thereby, activated. 63 biotin 64 B. Long term regulation of ACC  Prolonged consumption of high-carbohydrate diet increases acetyl CoA carboxylase synthesis and increases fatty acid synthesis.  High-fat diet or fasting decreases acetyl CoA carboxylase synthesis  Fatty acid synthase (FAS) complex: a multi enzyme complex reaction is the second major step in fatty acid biosynthesis  This enzyme system is more complex than acetyl-CoA carboxylase. 65 During fatty acid synthesis, two carbons from malonyl-CoA are added to a growing acyl chain that began with acetyl-CoA as a primer The series of reactions until the stage of palmitoyl- CoA is reached is catalyzed by FAS FAS is a dimeric protein with 2 identical subunits Each subunit has seven enzymatic activities and an acyl carrier protein (ACP) The ACP segment contains a phosphopantetheine residue The subunits of FAS are arranged in a “head-to-tail” manner 66 Each subunit has three domains: The first domain is responsible for the binding and condensation of acetyl and malonyl groups: contains acetyl transferase (AT), malonyl transferase (MT) and β-ketoacyl synthase (KS) The second domain reduces the intermediate produced by the first domain using NADP; contains ACP, β-ketoacyl reductase (KR), dehydratase (DH) and enoyl-CoA reductase (ER) The third domain contains thioesterase (TE) which releases the final product, palmitoyl CoA Growing acyl chains are transferred between the cysteinyl sulfhydryl group of the KS of one subunit and the phosphopantetheinyl sulfhydryl group of the ACP of the other subunit 67 Structure of FAS 68 69 70  Overview of Fatty Acid Synthesis 1/17/2025 71 72  This cycle of reactions is repeated 7 times, each time incorporating a two-carbon unit into a growing fatty acid chain.  Palmitate is liberated from the enzyme complex by the activity of a seventh enzyme in the complex, thioesterase.  All the carbons in the palmitic acid have passed through malonyl CoA except the two donated by the original acetyl CoA which are found at the methyl group end of the fatty acid. 73  The balance sheet of fatty acid synthesis The activation of acetyl-CoA: 7 acetyl-CoA + 7 CO2 + 7 ATP → 7 malonyl-CoA + 7 ADP + 7Pi Seven cycles of FAS reactions: acetyl-CoA + 7 malonyl-CoA + 14 NADPH + 14 H+→ palmitate + 7 CO2 + 14 NADP++ 8 CoA + 6 H2O 6 H2O because one water molecule is consumed by thioesterase Overall reaction: 8 acetyl-CoA + 7 ATP + 14 NADPH + 14 H+→ pal