2. Carbohydrate metabolism 2024.pdf

Carbohydrate metabolism BTech-MTech Biotechnology Semester III BT 2013 Biochemistry 1  Dietary polysaccharides and disaccharides undergo hydrolysis to monosaccharides For most humans, starch is the major...

Carbohydrate metabolism BTech-MTech Biotechnology Semester III BT 2013 Biochemistry 1  Dietary polysaccharides and disaccharides undergo hydrolysis to monosaccharides For most humans, starch is the major source of carbohydrates in the diet. Digestion begins in the mouth, where salivary α-amylase hydrolyzes the internal glycosidic linkages of starch, producing short polysaccharide fragments or oligosaccharides. In the stomach, salivary α-amylase is inactivated by the low pH. In the small intestine, α-amylase secreted by the pancreas continues the breakdown process. Pancreatic α-amylase yields mainly maltose and maltotriose (the di- and trisaccharides of α(1→4) glucose) and oligosaccharides called limit dextrins, fragments of amylopectin containing α(1 →6) branch points. Dietary glycogen has the same structure as starch, and its digestion proceeds by the same pathway. Disaccharides and oligosaccharides must be hydrolyzed to monosaccharides before entering cells. 2 Carbohydrate digestion in small intestine 3  Fate of glucose Glucose occupies a central position in the metabolism of plants, animals, and many microorganisms. Glucose is not only an excellent fuel, it is also a remarkably versatile precursor, capable of supplying a huge array of metabolic intermediates for biosynthetic reactions. In animals and vascular plants, glucose has three major fates: 1. It may be stored 2. Oxidized to pyruvate via glycolysis to provide ATP and metabolic intermediates 3. Oxidized via the pentose phosphate pathway to yield ribose 5-phosphate for nucleic acid synthesis and NADPH for reductive biosynthetic processes. 4  Glycolysis Glycolysis is an almost universal central pathway of glucose catabolism. In glycolysis a molecule of glucose is degraded in a series of enzyme-catalyzed reactions to yield two molecules of the three-carbon compound pyruvate. During the sequential reactions of glycolysis, some of the free energy released from glucose is conserved in the form of ATP and NADH. 5  Historical Perspective The fermentation of glucose to ethanol and CO2 by yeast (Winemaking and bread baking) has been a useful process since before the dawn of recorded history. 1854 to 1864 – Louis Pasteur established that fermentation is caused by microorganisms. 1897 – Eduard Buchner demonstrated that cell-free yeast extracts can also carry out this process. The philosophical shift that accompanied this discovery was announced by Jacques Loeb in 1906: “Through the discovery of Buchner, Biology was relieved of another fragment of mysticism. The splitting up of sugar into CO2 and alcohol is no more the effect of a “vital principle” than the splitting up of cane sugar by invertase. The history of this problem is instructive, as it warns us against considering problems as beyond our reach because they have not yet found their solution.” This was a major step in the development of biochemistry as a science. 6 In 1930s, the efforts of many investigators had come to fruition with the elucidation of the complete pathway of glycolysis. The work of three of these individuals, Gustav Embden, Otto Meyerhof, and Jacob Parnas, has been commemorated in that glycolysis is alternatively known as the Embden–Meyerhof–Parnas (EMP) pathway. Other major contributors to the elucidation of this pathway were Carl and Gerty Cori, Carl Neuberg, Robert Robison, Otto Warburg, Arthur Harden, William Young, Hans von Euler-Chelpin. 7 Glycolysis 8  ATP Formation in glycolysis Reaction catalyzed by Method of ATP formation ATP per Mol of glucose Glyceraldehyde 3-phosphate Respiratory chain oxidation of 2 5 dehydrogenase NADH Phosphoglycerate kinase Substrate level phosphorylation 2 Pyruvate kinase Substrate level phosphorylation 2 Consumption of ATP for reactions of hexokinase and –2 phosphofructokinase Net 7 9  Three possible catabolic fates of the pyruvate formed in glycolysis Pyruvate also serves as a precursor in many anabolic reactions, not shown here. 10  The anaerobic fate of pyruvate: Fermentation In aerobic conditions: NADH is passed into the mitochondria for reoxidation. In hypoxic and anaerobic conditions: NAD+ is replenished by the reduction of pyruvate in Lactic acid and Ethanol fermentation. 1. Lactic acid fermentation In muscle, particularly during vigorous activity when the demand for ATP is high and oxygen has been depleted, lactate dehydrogenase (LDH) catalyzes the oxidation of NADH by pyruvate to yield NAD+ and lactate. Some cell types such as erythrocytes have no mitochondria and thus cannot oxidize pyruvate to When lactate is produced in large quantities during CO2 even under aerobic conditions. vigorous muscle contraction, the acidification that results from ionization of lactic acid in muscle (can Much of the lactate is exported from the muscle drop the intramuscular pH from 7.0 to as low as cell via the blood to the liver, where it is 6.4) causes muscle fatigue and soreness. reconverted to glucose. 11 2. Ethanol fermentation Yeast and other microorganisms ferment glucose to ethanol and CO2, rather than to lactate. Pyruvate is converted to ethanol and CO2 in a two-step process: Ethanol is, of course, the active ingredient of wine and spirits; CO2 so produced leavens bread. From the point of view of the yeast, alcoholic fermentation has a practical benefit: Yeast employ ethanol as a kind of antibiotic to eliminate competing organisms. This is because yeast can grow in ethanol concentrations 12%, whereas few other organisms can survive in 5% ethanol. 12  Anaerobic glycolysis is used for rapid ATP production Pasteur effect: yeast consumes far more sugar when growing anaerobically than when growing aerobically. Fermentation results in the production of 2 ATPs per glucose, whereas oxidative phosphorylation yields 32 ATPs per glucose. The rate of ATP production by anaerobic glycolysis can be up to 100 times faster than that of oxidative phosphorylation. When tissues such as muscle are rapidly consuming ATP, they regenerate it almost entirely by anaerobic glycolysis. Skeletal muscles consist of two types of fibers: Slow-twitch (Type I) fibers: Designed to contract slowly and steadily Rich in mitochondria Obtain most of their ATP through oxidative phosphorylation Fast-twitch (Type II) fibers: Predominate in muscles capable of short bursts of rapid activity Nearly devoid of mitochondria Obtain nearly all of their ATP through anaerobic glycolysis 13  Regulation of Glycolysis 1. Hexokinase and blood glucose homeostasis Isozymes of hexokinase: Extrahepatic tissues (Muscle) : Hexokinase I – III (Km ≈ 1 mM) Liver, pancreas : Hexokinase IV (glucokinase) (Km ≈ 10 mM) Extrahepatic tissues consumes glucose for energy Liver maintains blood glucose homeostasis During fed state (Blood glucose > 5 mM) § In extrahepatic tissues, hexokinases I–III operates normal to provide G6P to metabolism. § In liver, hexokinase IV converts it to G6P to store the glucose as glycogen, thereby reduce the blood glucose level. During a fast (Blood glucose < 5 mM) § In extrahepatic tissues, hexokinases I–III operates normal as the blood glucose concentration is higher than their Km. § Hexokinase IV is inhibited (1) due to glucose concentration is much lower than the Km of glucokinase and (2) by a inhibitory protein. So, 14 the liver does not compete with other organs for the scarce glucose. 2. Phosphofructokinase Is under complex allosteric regulation Glucose 6-phosphate can flow either into glycolysis or through any of several other pathways, including glycogen synthesis and the pentose phosphate pathway. The irreversible reaction catalyzed by PFK is the step that commits glucose to glycolysis. In addition to its substrate-binding sites, this PFK complex enzyme has several regulatory sites at which allosteric activators or inhibitors bind. Allosteric inhibitors of PFK: ATP, Citrate, PEP Allosteric activators of PFK: ADP, AMP, cAMP, FBP, F2,6P, NH4+, Pi 15 3. Pyruvate kinase is under both allosteric and hormonal control (active) The inactivation of liver isozyme by phosphorylation slows the use of glucose as a fuel in liver, sparing it for export to the brain and other organs. 16 Feeder Pathways for Glycolysis: Entry of dietary glycogen, starch, disaccharides, and hexoses into the glycolysis 17 Citric acid cycle/Tricarboxylic acid (TCA) cycle/ Krebs cycle Most eukaryotic cells and many bacteria, which live under aerobic conditions, oxidize their organic fuels to CO2 and water. This aerobic phase of catabolism is called respiration – the molecular processes by which cells consume O2 and produce CO2 are termed as cellular respiration. Cellular respiration occurs in three major stages: 1. Organic fuel molecules are oxidized to yield two-carbon fragments, acetyl-CoA. 2. The citric acid cycle oxidizes acetyl groups to CO2; the energy released is conserved in the reduced electron carriers NADH and FADH2. 3. These NADH and FADH2 are oxidized, giving up protons (H+) and electrons. The electrons are transferred to O2 via a respiratory chain; the large amount of energy released is conserved in the form of ATP, by a process called oxidative phosphorylation.  Production of Acetyl-CoA (Activated Acetate) Under aerobic conditions pyruvate, which enters the mitochondrion via a specific pyruvate-H+ symport, is converted to acetyl- CoA by pyruvate dehydrogenase. The PDH complex is a classic example of a multienzyme complex. Five cofactors, four derived from vitamins, participate in the reaction mechanism. Mutations in the genes for the subunits of the PDH complex, or a dietary thiamine deficiency (Beriberi), can have severe consequences. Beriberi is characterized by loss of neural function; brain, usually obtains all its energy from the aerobic oxidation of glucose..  Substrate channeling The pyruvate dehydrogenase complex consists of three distinct enzymes – In substrate channeling, intermediates never leave the enzyme surface 20  Bovine PDH cryoelectron micrograph Bovine PDH – 60 monomers (20 E1, 20 E2, 20 E3) Azotobacter vinelandii PDH Bacillus stearothermophilus PDH 24 monomers (8 E1, 8 E2, 8 E3) 60 monomers (20 E1, 20 E2, 20 E3) Oxidation of glucose yields 32 ATP under aerobic conditions, but only 2 when O2 is absent Reaction catalyzed by Method of ATP formation ATP per glucose Glycolysis Glyceraldehyde 3-phosphate Oxidative phosphorylation of 2 NADH 5 dehydrogenase Phosphoglycerate kinase Substrate level phosphorylation 2 Pyruvate kinase Substrate level phosphorylation 2 Consumption of ATP for reactions of hexokinase and phosphofructokinase –2 Net 7 Citric acid cycle Pyruvate dehydrogenase Oxidative phosphorylation of 2 NADH 5 Isocitrate dehydrogenase Oxidative phosphorylation of 2 NADH 5 α-Ketoglutarate dehydrogenase Oxidative phosphorylation n of 2 NADH 5 Succinyl Co-A synthetase Substrate level phosphorylation 2 Succinate dehydrogenase Oxidative phosphorylation n of 2 FADH2 3 Malate dehydrogenase Oxidative phosphorylation of 2 NADH 5 Net 25 Total ATP per glucose under aerobic conditions 32 Total per glucose under anaerobic conditions 2 Regulation of the citric acid cycle  The amphibolic nature of the citric acid cycle Ordinarily one thinks of a metabolic pathway as being either catabolic with the release (and conservation) of free energy, or anabolic with a requirement for free energy. The citric acid cycle is catabolic because it involves degradation and is a major free energy conservation system in most organisms, and anabolic because several biosynthetic pathways utilize citric acid cycle intermediates as starting materials. The citric acid cycle is therefore called amphibolic pathway. This pathway is the hub of intermediary metabolism: End products of many catabolic processes feed into the cycle to serve as fuels and, intermediates are drawn out of the cycle to be used as precursors in a variety of biosynthetic pathways. The citric acid cycle is truly at the center of metabolism. Role of the citric acid cycle in anabolism  Anaplerotic reactions replenish citric acid cycle intermediates As intermediates of the citric acid cycle are removed to serve as biosynthetic precursors, they are replenished by anaplerotic reactions. Degradative pathways also generate citric acid cycle intermediates: 1. Oxidation of odd-chain fatty acids leads to the production of succinyl-CoA. 2. Breakdown of amino acids isoleucine, methionine, and valine also leads to the production of succinyl-CoA. 3. Transamination and deamination of amino acids lead to the production of α-ketoglutarate and oxaloacetate. These reactions are reversible and, depending on metabolic demand, serve to remove or replenish these citric acid cycle intermediates.  Glycogen metabolism Higher organisms protect themselves from potential fuel shortage by polymerizing excess glucose for storage as high molecular mass glucans (starch in plants, glycogen in animals). In vertebrates, glycogen is found primarily in the liver (upto 10% glycogen by weight; 100-120g) and skeletal muscle (1–2% glycogen by weight). Muscle glycogen can be exhausted in less than an hour during vigorous activity. Liver glycogen serves as a reservoir of glucose for other tissues (12 to 24 h supply) when dietary glucose is not available (between meals or during a fast). Why does the body go to such metabolic effort to use glycogen for energy storage when fat, which is far more abundant in the body, seemingly serves the same purpose? The answer is threefold: 1. Muscles cannot mobilize fat as rapidly as they can glycogen. 2. The fatty acid residues of fat cannot be metabolized anaerobically. 3. Animals cannot convert fatty acids to glucose; so fat metabolism alone cannot adequately maintain essential blood glucose levels. 28 Glycogen structure 29 29  Glycogenolysis (Glycogenlysis, Glycogen breakdown) In muscle: The need for ATP results in the conversion of glycogen to glucose-6-phosphate (G6P) for entry into glycolysis. In liver: Low blood glucose concentration triggers glycogen breakdown to G6P. Then, G6P is hydrolyzed to glucose and released into the bloodstream to reverse this situation. The glucose units of glycogen enter the glycolysis through the action of three enzymes: 1. Glycogen phosphorylase 2. Glycogen debranching enzyme 3. Phosphoglucomutase. 30 Glycogen breakdown 31 Glycogenesis (Glycogen synthesis) 32  The branching of glycogen Branches are formed by transferring a 7-residue terminal segment from an α(1→4) -linked glucan chain to the C6 -OH group of a glucose residue on the same or another chain. 33  Glycogenin primes the initial sugar residues in glycogen Glycogen synthase cannot simply link together two glucose residues; it can only extend an already existing (α1→4)-linked glucan chain of at least 8 residues. How, then, is glycogen synthesis initiated? The first step in glycogen synthesis is the self-catalyzed attachment of a glucose residue to the Tyr194 OH Glycogenin structure (Red: The substrate, UDP- group of a 332-residue homodimeric protein named glucose; Green: Mn2+ ion bound to the phosphates glycogenin. of UDP-glucose; Orange: Asp162 Glycogenin further extends the glucan chain by up to ≈9 additional UDP–glucose-supplied residues, forming a “primer” for the initiation of glycogen synthesis. At this point, glycogen synthase takes over, further extending the glycogen chain. Glycogenin remains buried within the particle, covalently attached to the single reducing end of the glycogen molecule. 34 Coordinated regulation of glycogen synthesis and breakdown Glycogen phosphorylase is regulated is regulated allosterically and hormonally 35 Cascade mechanism of epinephrine and glucagon action Glucose transporters GLUT1 Widely distributed in fetal tissues. Adults: expressed at highest levels in erythrocytes and barrier tissues (blood–brain barrier). GLUT2 Expressed by renal tubular cells, small intestinal epithelial cells, liver cells and pancreatic beta cells. Bidirectional transporter. GLUT3 Expressed mostly in neurons and in the placenta. GLUT4 Expressed in adipose tissues and striated muscle. Insulin-regulated glucose transporter. 36 Glycogen synthase is also regulated by phosphorylation and dephosphorylation 37 Regulation of carbohydrate metabolism in the hepatocyte 38 Much of the metabolism of glycogen was discovered between about 1925 and 1950 by the Carl F. Cori and Gerty T. Cori. Carl and Gerty Cori shared the Nobel Prize in Physiology or Medicine in 1947 with Bernardo Houssay of Argentina, who was cited for his studies of hormonal regulation of carbohydrate metabolism. At least six scientists who trained with the Coris became Nobel laureates: Arthur Kornberg – for DNA synthesis, 1959 Severo Ochoa – for RNA synthesis, 1959 Luis Leloir – for the role of sugar nucleotides in polysaccharide synthesis, 1970 Earl Sutherland – for the discovery of cAMP in the regulation of carbohydrate metabolism, 1971 Christian de Duve – for subcellular fractionation, 1974 Edwin Krebs – for the discovery of phosphorylase kinase, 1991 39 Glycogen storage diseases of humans 40  Type I: Glucose-6-phosphatase deficiency (von Gierke’s Disease) (1 in 50,000 births) Glucose-6-Phosphatase deficiency results in: – an increase of intracellular G6P – large accumulation of glycogen of in the liver and kidney (G6P inhibits glycogen phosphorylase and activates glycogen synthase) – inability to increase blood glucose in response to glucagon or epinephrine Symptoms Massive liver enlargement and severe hypoglycemia after a few hours’ fast Treatment – Drug-induced inhibition of glucose uptake by the liver to increase blood glucose – Continuous intragastric feeding overnight to increase blood glucose – Oral administration of uncooked corn starch – Surgical transposition of the portal vein (to allow this glucose-rich blood to reach peripheral tissues before it reaches the liver) – Liver transplantation has also been successful in the few patients in which tried. – A gene therapy protocol is being developed to correct type I glycogen storage disease. 41  Type II: α-1,4-Glucosidase deficiency (Pompe’s disease) (1 in 40,000 births) This is the most devastating glycogen storage disease. It results in a large accumulation of glycogen in the lysosomes of all cells. Causes death by cardiorespiratory failure, usually before the age of 1 year. α-1,4-glucosidase occurs in lysosomes, where it functions to hydrolyze the disaccharide maltose and linear oligosaccharides, as well as the outer branches of glycogen, thereby yielding free glucose. However, this pathway of glycogen metabolism is not quantitatively important. The reason that lysosomes normally take up and degrade glycogen granules is unknown. 42  Gluconeogenesis In mammals, some tissues depend almost completely on glucose for their metabolic energy. Eg: In human, brain and nervous system, erythrocytes, testes, renal medulla, and embryonic tissues. The human brain alone requires about 120 g of glucose each day. However, the supply of glucose from glycogen is not always sufficient; between meals and during longer fasts, or after vigorous exercise, glycogen is depleted. For these times, organisms need a method for synthesizing glucose from noncarbohydrate precursors. This is accomplished by a pathway called gluconeogenesis (formation of new sugar), which converts pyruvate and related three- and four-carbon compounds to glucose. Gluconeogenesis occurs in all animals, plants, fungi, and microorganisms; the reactions are essentially the same in all tissues and all species. In mammals, gluconeogenesis takes place mainly in the liver, and to a lesser extent in renal cortex. The glucose produced passes into the blood to supply other tissues. 43 Carbohydrate synthesis from simple precursors 44 Opposing pathways of glycolysis and gluconeogenesis Gluconeogenesis requires metabolite transport between mitochondria and cytosol 45 Pathways converting lactate, pyruvate, and citric acid cycle intermediates to oxaloacetate 46  Regulation of gluconeogenesis If both glycolysis and gluconeogenesis were to proceed in an uncontrolled manner, the net effect would be a futile cycle wastefully hydrolyzing ATP and GTP. So, these pathways are reciprocally regulated. In the fed state, when the blood glucose level is high, the liver is geared toward fuel conservation by: (a) glycogen synthesis, and (b) activating glycolytic pathway to break down glucose to acetyl-CoA for fatty acid biosynthesis and fat storage. In the fasted state liver maintains the blood glucose level by (a) glycogen breakdown, and (b) reversing the flux through glycolysis toward gluconeogenesis. The rate and direction of glycolysis and gluconeogenesis are controlled at the reactions catalyzed by (1) Hexokinase/Glucose-6-phosphatase (2) PFK/FBPase (3) Pyruvate kinase/Pyruvate carboxylase–PEPCK 47  Regulators of gluconeogenic enzyme activity Liver pyruvate kinase is inhibited both allosterically by alanine (a pyruvate precursor) and by phosphorylation. Then the pathway flow toward G6P, which is converted to glucose for export to muscle and brain. Muscle pyruvate kinase, an isozyme of the liver enzyme, is not subject to these controls. 48  The Cori cycle Muscle produce lactate when ATP demand exceeds oxidative flux. The lactate is transferred, via the bloodstream, to the liver, where it is reconverted to pyruvate by LDH and then to glucose by gluconeogenesis. The resynthesized glucose is returned to the muscle, where it is stored as glycogen. Thus, through the intermediacy of the bloodstream, liver and muscle participate in a metabolic cycle known as the Cori cycle in honor of Carl and Gerty Cori, who first described it. In this process liver ATP is used to resynthesize glucose from lactate produced in muscle. The ATP utilized by the liver is regenerated by oxidative phosphorylation. After vigorous exertion, it often takes at least 30 min for all of the lactate so produced to be converted to glycogen and the oxygen consumption rate to return to its resting level, a phenomenon known as oxygen debt. 49 Difference in the regulation of carbohydrate metabolism in liver and muscle 50  Pentose phosphate pathway (or) Hexose monophosphate (HMP) shunt (or) Phosphogluconate pathway ATP is the cell’s “energy currency”; cells also have a second currency, reducing power. Many endergonic reactions, notably the reductive biosynthesis of fatty acids and cholesterol, as well as photosynthesis, require NADPH in addition to ATP. NADH – Participates in utilizing the free energy of metabolite oxidation to synthesize ATP. Cells maintain [NAD+]/[NADH] ratio near 1000, which favors metabolite oxidation. NADPH – Involved in utilizing the free energy of metabolite oxidation for endergonic reductive biosynthesis. Cells maintain [NADP+]/[NADPH] ratio near 0.01, which favors metabolite reduction. NADPH is generated by the oxidation of G6P via the pentose phosphate pathway, an alternative pathway to glycolysis. The pathway also produces ribose-5-phosphate (R5P), an essential precursor in nucleotide biosynthesis. Tissues most heavily involved in fatty acid synthesis (liver, adipose, lactating mammary gland) or cholesterol and steroid hormones synthesis (liver, adrenal gland, gonads) are rich in pentose phosphate pathway enzymes. Indeed, some 30% of the glucose oxidation in liver occurs via the pentose phosphate pathway. 52 Pentose phosphate pathway  Control of the pentose phosphate pathway Flux through the oxidative pentose phosphate pathway and thus the rate of NADPH production is controlled by the rate of the glucose-6-phosphate dehydrogenase reaction. The activity of G6PD enzyme is regulated by the NADP+ concentration (substrate availability). When the need for NADPH exceeds R5P: The transaldolase and transketolase reactions serve to convert excess R5P to glycolytic intermediates. The resulting GAP and F6P can be consumed through glycolysis or recycled by gluconeogenesis to form G6P. When the need for R5P exceeds NADPH: F6P and GAP can be diverted from the glycolytic pathway for use in the synthesis of R5P by reversal of the transaldolase and transketolase reactions.  Glucose-6-phosphate dehydrogenase (G6PD) deficiency G6PD deficiency is an X-linked recessive hereditary disease. Most G6PD deficient individuals are asymptomatic; results in severe hemolytic anemia on infection or on the administration of certain drugs including the antimalarial agent primaquine. G6PD deficiency is closely linked to favism, a disorder characterized by a hemolytic reaction to consumption of fava beans. Fava beans contain small quantities of alkaloids such as fovicine, isouramil and covicine which stimulate peroxide formation, thereby increasing the demand for NADPH to a level that mutant cells cannot meet. In favism, erythrocytes begin to lyse 24 to 48 h after ingestion of the beans, releasing free hemoglobin into the blood. Jaundice and sometimes kidney failure can result. Over 400 million people are deficient in G6PD, which makes this condition the most common human enzymopathy. 400 G6PD variants have been reported and at least 140 of them have been characterized at the molecular level. Variants occur with high incidence: 1. Type A deficiency (10% of the normal G6PD activity) 11% incidence among African Americans Most common form of G6PD deficiency in sub-Saharan Africa 2. “Mediterranean” variant Found throughout the Mediterranean and Middle East regions Occurs in 65% of Kurdish Jews (the population with the highest incidence) In vitro studies indicate that erythrocytes with G6PD deficiency are less suitable hosts for plasmodia than are normal cells. Thus, like the sickle-cell trait, a defective G6PD confers a selective advantage on individuals living where malaria is endemic. This is presumably because the parasite requires the products of the pentose phosphate pathway and/or because the erythrocyte is lysed before the parasite has had a chance to mature. Alcohol metabolism 57 The images were adapted from: 1. Lehninger Principles of Biochemistry, fifth edition. Authors: Nelson DL and Cox MM. W. H. Freeman and company. 2008. 2. Biochemistry, fourth edition. Authors: Voet G & Voet JG. John Wiley and Sons, Inc. 2011. 3. Harper’s Illustrated Biochemistry, twenty-sixth edition. Authors: Murray RK, Granner DK, Mayes PA, Rodwell VW. McGraw-Hill Companies, Inc., 2003. 4. Lippincott’s Illustrated Reviews: Biochemistry, sixth edition. Authors: Ferrier DR, Lippincott Williams & Wilkins, a Wolters Kluwer business, 2014.