Intertissue Relationships in the Metabolism of Amino Acids

• The body maintains a large free amino acid pool in the blood, even during fasting, allowing tissues continuous access to these building blocks.
• Amino acids are used for gluconeogenesis by the liver, as a fuel source for the gut, and as neurotransmitter precursors in the nervous system. They are also required by all organs for protein synthesis.
• During an overnight fast and during hypercatabolic states, degradation of labile protein (primarily from skeletal muscle) is the major source of free amino acids.
• The liver is the major site of urea synthesis. Nitrogen from other tissues travels to the liver in the form of glutamine and alanine.
• Branched-chain amino acids are oxidized primarily in the skeletal muscle.
• Glutamine in the blood serves a number of roles:
  • The kidney utilizes the ammonium ion carried by glutamine for excretion in the urine to act as a buffer against acidotic conditions.
  • The kidney and the gut utilize glutamine as a fuel source.
  • All tissues utilize glutamine for protein synthesis.
• The body can enter a catabolic state characterized by negative nitrogen balance under the following conditions:
  • Sepsis (any of various pathogenic organisms or their toxins in the blood or tissues)
  • Trauma
  • Injury
  • Burns
• The negative nitrogen balance results from increased net protein degradation in skeletal muscle, brought about by release of glucocorticoids. The released amino acids are used for protein synthesis and cell division in cells involved in the immune response and wound healing.

Purine and Pyrimidine Metabolism

• Purine and pyrimidine nucleotides can both be synthesized from scratch (de novo) or salvaged from existing bases.
• De novo purine synthesis is complex, requiring 11 steps and 6 molecules of adenosine triphosphate for every purine synthesized. Purines are initially synthesized in the ribonucleotide form.
• The precursors for de novo purine synthesis are glycine, ribose 5-phosphate, glutamine, aspartate, carbon dioxide, and N¹⁰-formyltetrahydrofolate.
• The initial purine ribonucleotide synthesized is inosine monophosphate. Adenosine monophosphate and guanosine monophosphate are each derived from inosine monophosphate.
• Since de novo purine synthesis requires a large amount of energy, purine nucleotide salvage pathways exist such that free purine bases can be converted to nucleotides.
• Mutations in purine salvage enzymes are associated with severe diseases, such as Lesch-Nyhan syndrome and severe combined immunodeficiency disease.
• Pyrimidine bases are initially synthesized as the free base and then converted to nucleotides.
• Aspartate and cytoplasmic carbamoyl phosphate are the precursors for pyrimidine ring synthesis.
• The initial pyrimidine nucleotide synthesized is orotate monophosphate, which is converted to uridine monophosphate. The other pyrimidine nucleotides will be derived from a uracil-containing intermediate.
• Deoxyribonucleotides are derived by reduction of ribonucleotides, as catalyzed by ribonucleotide reductase. The regulation of ribonucleotide reductase is complex.
• Degradation of purine containing nucleotides results in uric acid production, which is eliminated in the urine. Elevated uric acid levels in the blood lead to gout.

Tetrahydrofolate, Vitamin B12, and S-Adenosylmethionine

• One-carbon groups at lower oxidation states than carbon dioxide (which is carried by biotin) are transferred by reactions involving tetrahydrofolate, vitamin B₁₂, and S-adenosyl methionine.
• Tetrahydrofolate is produced from the vitamin folate and obtains one-carbon units from serine, glycine, histidine, formaldehyde, and formic acid.
• The carbon attached to tetrahydrofolate can be oxidized or reduced, thus producing a number of different forms of tetrahydrofolate. However, once a carbon has been reduced to the methyl level, it cannot be reoxidized.
• The carbons attached to tetrahydrofolate are known collectively as the one-carbon pool.
• The carbons carried by folate are used in a limited number of biochemical reactions but are very important in forming deoxythymidine monophosphate and the purine rings.
• Vitamin B₁₂ participates in two reactions in the body: conversion of L-methylmalonyl coenzyme A to succinyl coenzyme A and the conversion of homocysteine to methionine.
• S-adenosyl methionine, formed from adenosine triphosphate and methionine, transfers the methyl group to precursors forming a variety of methylated compounds.
• Both vitamin B₁₂ and methyl tetrahydrofolate are required in methionine metabolism; a deficiency of vitamin B₁₂ leads to overproduction and trapping of folate in the methyl form, leading to a functional folate deficiency. Such deficiencies can lead to
  • Megaloblastic anemia
  • Neural tube defects in newborn

Synthesis and Degradation of Amino Acids and Amino Acid Derived Products

• Humans can synthesize only 11 of the 20 amino acids required for protein synthesis; the other 9 are considered essential amino acids for the diet.
• Amino acid metabolism to a large extent utilizes the cofactors pyridoxal phosphate, tetrahydrobiopterin, and tetrahydrofolate.
  • Pyridoxal phosphate is primarily required for transamination reactions.
  • Tetrahydrobiopterin is required for ring hydroxylation reactions.
  • Tetrahydrofolate is required for one-carbon metabolism (discussed further in Chapter 33).
• The nonessential amino acids can be synthesized from glycolytic intermediates (serine, glycine, cysteine and pyruvate), tricarboxylic acid cycle intermediates (aspartate, asparagine, glutamate, glutamine, proline, arginine, and ornithine), or from existing amino acids (tyrosine from phenylalanine).
• When amino acids are degraded, the nitrogen is converted to urea, and the carbon skeletons are classified as either glucogenic (a precursor of glucose) or ketogenic (a precursor of ketone bodies).
• Defects in amino acid degradation pathways can lead to disease.
  • Glycine degradation can lead to oxalate production, which may lead to one class of kidney stone formation.
  • Defects in methionine degradation can lead to hyper homocysteinemia, which has been linked to blood clotting disorders and heart disease.
  • A defect in branched-chain amino acid degradation leads to maple syrup urine disease, which has severe neurological consequences.
  • Defects in phenylalanine and tyrosine degradation lead to phenylketonuria, alcaptonuria, and albinism.
• Amino acids are also the precursors for the small nitrogen-containing neurotransmitters, such as the catecholamines, serotonin, and histamine.
• Glycine is required for the biosynthesis of heme. Mutations in enzymes involved in heme biosynthesis give rise to a class of diseases known as the porphyrias.

Fate of Amino Acid Nitrogen: Urea Cycle

• Amino acid catabolism will generate urea, a nontoxic carrier of nitrogen atoms.
• Urea synthesis occurs in the liver. The amino acids alanine and glutamine will carry amino acid nitrogen from peripheral tissues to the liver.
• Key enzymes involved in nitrogen disposal are transaminases, glutamate dehydrogenase, and glutaminase.
• The urea cycle consists of four steps and incorporates a nitrogen from ammonia and one from aspartate into urea.
• Disorders of the urea cycle lead to hyperammonemia, a condition toxic to the nervous system, health, and development.

Protein Digestion and Amino Acid Absorption

• Proteases (proteolytic enzymes) break down dietary proteins into peptides and then their constituent amino acids in the stomach and intestine.
• Pepsin initiates protein breakdown in the stomach.
• Upon entering the small intestine, inactive zymogens secreted from the pancreas are activated to continue protein digestion.
• Enzymes produced by the intestinal epithelial cells are also required to fully degrade proteins.
• The amino acids generated by proteolysis in the intestinal lumen are transported into the intestinal epithelial cells, from which they enter the circulation for use by the tissues.
• Transport systems for amino acids are similar to transport systems for monosaccharides; both facilitative and active transport systems exist.
• There are a large number of overlapping transport systems for amino acids in cells.
• Protein degradation (turnover) occurs continuously in all cells.
• Proteins can be degraded by lysosomal enzymes (cathepsins).
• Proteins are also targeted for destruction by being covalently linked to the small protein ubiquitin.
• The ubiquitin-tagged proteins interact with the proteosome, a large complex designed to degrade proteins to small peptides in an adenosine triphosphate–dependent process.
• Amino acids released from proteins during turnover can be used for the synthesis of new proteins, for energy generation, or for gluconeogenesis.

Integration of Carbohydrate and Lipid Metabolism

• Three key controlling elements determine whether a fuel is metabolized or stored: hormones, concentration of available fuels, and energy needs of the body.
• Key intracellular enzymes are generally regulated by allosteric activation and inhibition, by covalent modification, by transcriptional control, and by degradation.
• Regulation is complex in order to allow sensitivity and feedback to multiple stimuli so that an exact balance can be maintained between synthesis of a product and need for the product.
• The insulin/glucagon ratio is responsible for the hormonal regulation of carbohydrate and lipid metabolism.

Cholesterol Absorption, Synthesis, Metabolism, and Fate

• Cholesterol regulates membrane fluidity and is a precursor of bile salts, steroid hormones (such as estrogen and testosterone), and vitamin D.
• Cholesterol, because of its hydrophobic nature, is transported in the blood as a component of lipoproteins.
• Within the lipoproteins, cholesterol can appear in its unesterified form in the outer shell of the particle or as cholesterol esters in the core of the particle.
• De novo cholesterol synthesis requires acetyl coenzyme A as a precursor, which is initially converted to β-hydroxy-β-methylglutaryl-coenzyme A (HMG-CoA). The cholesterol synthesized in this way is packaged, along with triglyceride, into very low density lipoprotein in the liver and released into circulation.
• The conversion of HMG-CoA to mevalonic acid, catalyzed by HMG-CoA reductase, is the regulated and rate-limiting step of cholesterol biosynthesis.
• In the circulation, the triglycerides in very low density lipoproteins are digested by lipoprotein lipase, which converts the particle to intermediate-density lipoprotein and then to low-density lipoprotein.
• Intermediate- and low-density lipoprotein bind specifically to receptors on the liver cell, are internalized, and the particle components recycled.
• A third lipoprotein particle, high-density lipoprotein, functions to transfer apolipoprotein E and apolipoprotein C-II to nascent chylomicrons and nascent very low density lipoprotein.
• High-density lipoprotein also participates in reverse cholesterol transport, the movement of cholesterol from cell membranes to the high-density lipoprotein particle, which returns the cholesterol to the liver.
• Atherosclerotic plaques are associated with elevated levels of blood cholesterol levels. High levels of low-density lipoprotein are more strongly associated with the generation of atherosclerotic plaques, whereas high levels of high-density lipoprotein are protective because of their participation in reverse cholesterol transport.

Synthesis of Fatty Acids, Triacylglycerols, Eicosanoids, and the Major Membrane Lipids

• Fatty acids are synthesized mainly in the liver, primarily from glucose.
• Glucose is converted to pyruvate via glycolysis, which enters the mitochondrion and forms both acetyl coenzyme A and oxaloacetate, which then forms citrate.
• The newly synthesized citrate is transported to the cytosol, where it is cleaved to form acetyl coenzyme A, which is the source of carbons for fatty acid biosynthesis.
• Two enzymes, acetyl coenzyme A carboxylase (the key regulatory step) and fatty acid synthase, produce palmitic acid (16 carbons, no double bonds) from acetyl coenzyme A. After activation to palmitoyl coenzyme A, the fatty acid can be elongated or desaturated (adding double bonds) by enzymes in the endoplasmic reticulum.
• The eicosanoids (prostaglandins, thromboxanes and leukotrienes) are potent regulators of cellular function and are derived from polyunsaturated fatty acids containing 20 carbon atoms.
• Fatty acids are used to produce triacylglycerols (for energy storage) and glycerol phospholipids and sphingolipids (for structural components of cell membranes).
• Liver-derived triacylglycerol is packaged with various apolipoproteins and secreted into the circulation as very low density lipoprotein.
• As with dietary chylomicrons, lipoprotein lipase in the capillaries of adipose tissue, muscle, and the lactating mammary gland digests the triacylglycerol of very low density lipoprotein, forming fatty acids and glycerol.
• Glycerophospholipids, synthesized from fatty acyl CoA and glycerol 3-phosphate, are all derived from phosphatidic acid. Various head groups are added to phosphatidic acid to form the mature glycerol phospholipids.
• Phospholipid degradation is catalyzed by phospholipases.
• Sphingolipids are synthesized from sphingosine, which is derived from palmitoyl coenzyme A and serine. Glycolipids, such as cerebrosides, globosides, and gangliosides, are sphingolipids.
• The sole sphingosine-based phospholipid is sphingomyelin.

Digestion and Transport of Dietary Lipids

• Triacylglycerols are the major fat source in the human diet.
• Lipases (lingual lipase in the saliva and gastric lipase in the stomach) perform limited digestion of triacylglycerol prior to entry into the intestine.
• Cholecystokinin is released by the intestine as food enters, which signals the gallbladder to release bile acids and the exocrine pancreas to release digestive enzymes.
• Within the intestine, bile salts emulsify fats, which increases their accessibility to pancreatic lipase and colipase.
• Triacylglycerols are degraded to form free fatty acids and 2-monoacylgylcerol by pancreatic lipase and colipase.
• Dietary phospholipids are hydrolyzed by pancreatic phospholipase A2 in the intestine.
• Dietary cholesterol esters (cholesterol esterified to a fatty acid) are hydrolyzed by pancreatic cholesterol esterase in the intestine.
• Micelles, consisting of bile acids and the products of fat digestion, form within the intestinal lumen and interact with the enterocyte membrane. Lipid-soluble components diffuse from the micelle into the cell.
• Bile salts are resorbed farther down the intestinal tract and returned to the liver by the enterohepatic circulation.
• The intestinal epithelial cells resynthesize triacylglycerol and package them into chylomicrons for release into the circulation.
• Once in circulation the nascent chylomicrons interact with high-density lipoprotein particles and acquire two additional protein components; apolipoproteins C-II and E.
• ApoCII activates lipoprotein lipase on capillary endothelium of muscle and adipose tissue, which digests the triglycerides in the chylomicron. The fatty acids released from the chylomicron enter the muscle for energy production or the fat cell for storage. The glycerol released is metabolized only in the liver.
• As the chylomicron loses triglyceride, its density increases, and it becomes a chylomicron remnant. Chylomicron remnants are removed from circulation by the liver through specific binding of the remnant to apolipoprotein E receptors on the liver membrane.
• Once in the liver the remnant is degraded, and the lipids are recycled.

Gluconeogenesis and Maintenance of Blood Glucose Levels

• The process of glucose production is termed gluconeogenesis. Gluconeogenesis occurs primarily in the liver.
• The major precursors for glucose production are lactate, glycerol, and amino acids.
• The gluconeogenic pathway utilizes the reversible reactions of glycolysis, plus additional reactions to bypass the irreversible steps.
  • Pyruvate carboxylase (pyruvate to oxaloacetate) and phosphoenolpyruvate carboxykinase (oxaloacetate to phosphoenolpyruvate) bypass the pyruvate kinase step.
  • Fructose 1,6-bisphosphatase (fructose 1,6-bisphosphate to fructose 6-phosphate) bypasses the phosphofructokinase-1 step.
  • Glucose 6-phosphatase (glucose 6-phosphate to glucose) bypasses the glucokinase step.
• Gluconeogenesis and glycogenolysis are carefully regulated such that blood glucose levels can be maintained at a constant level during fasting. The regulation of triglyceride metabolism is also linked to the regulation of blood glucose levels.

Synthesis of Glycosides, Lactose, Glycoproteins, Glycolipids and Proteoglycans

• Reactions between sugars or the formation of sugar derivatives utilize sugars activated by attachment to nucleotides (a nucleotide sugar).
• UDP-glucose and UDP-galactose are substrates for many glycosyltransferase reactions.
• Lactose is formed from UDP-galactose and glucose.
• UDP-glucose is oxidized to UDP-glucuronate, which forms glucuronide derivatives of various hydrophobic compounds, making them more readily excreted in urine or bile than the parent compound.
• Glycoproteins and glycolipids contain various types of carbohydrate residues.
• The carbohydrates in glycoproteins can be either O-linked or N-linked and are synthesized in the endoplasmic reticulum and Golgi apparatus.
• For O-linked carbohydrates, the carbohydrates are added sequentially (via nucleotide sugar precursors), beginning with a sugar linked to the hydroxyl group of the amino acid side chains of serine or threonine.
• For N-linked carbohydrates, the branched carbohydrate chain is first synthesized on dolichol phosphate and then transferred to the amide nitrogen of an asparagine residue of the protein.
• Glycolipids belong to the class of sphingolipids, synthesized from nucleotide sugars that add carbohydrate groups to the base ceramide.
• Defects in the degradation of glycosphingolipids leads to a class of lysosomal diseases known as the sphingolipidoses.
• Proteoglycans consist of a core protein covalently attached to many long, linear chains of glycosaminoglycans, which contain repeating disaccharide units. Proteoglycans are synthesized in the endoplasmic reticulum and Golgi complex.
• The major carbohydrates in glycosaminoglycans are a hexosamine and uronic acid, along with sulfated carbohydrates.
• Failure to appropriately degrade proteoglycans within the lysosome leads to a set of disorders known as the mucopolysaccharidoses.

Pathways of Sugar and Alcohol Metabolism

Pathways of Sugar and Alcohol Metabolism: Fructose, Galactose, Pentose Phosphate Pathway, and Ethanol Metabolism:

• Fructose is ingested principally as the monosaccharide or as part of sucrose. Fructose metabolism generates fructose 1-phosphate, which is converted to intermediates of the glycolytic pathway.
• Galactose is ingested principally as lactose, which is converted to glucose and galactose in the intestine. Galactose metabolism generates first galactose 1-phosphate, which is converted to uridine diphosphate (UDPgalactose). The end product is glucose 1-phosphate, which is isomerized to glucose 6-phosphate, which enters glycolysis.
• The energy yield through glycolysis for both fructose and galactose is the same as for glucose metabolism.
• The pentose phosphate pathway consists of both oxidative and nonoxidative reactions.
• The oxidative steps of the pentose phosphate pathway generate NADPH and ribulose 5-phosphate from glucose 6-phosphate.
  • Ribulose 5-phosphate is converted to ribose 5-phosphate for nucleotide biosynthesis.
  • NADPH is utilized as reducing power for biosynthetic pathways.
• The nonoxidative steps of the pentose phosphate pathway reversibly convert five-carbon sugars to fructose 6-phosphate and glyceraldehyde 3-phosphate.
• Ethanol is metabolized to acetate primarily in the liver, generating NADH.
• The enzymes involved in ethanol metabolism are alcohol and aldehyde dehydrogenases.
• High or chronic ethanol ingestion induces a microsomal ethanol oxidizing system composed of cytochrome P450 enzymes in the endoplasmic reticulum.
• Acute effects of ethanol ingestion arise principally from the generation of NADH, which increases the NADH/NAD⁺ ratio of the liver. This leads to the following:
  • Inhibition of fatty acid oxidation
  • Inhibition of ketogenesis
  • Lactic acidosis
  • Hypoglycemia
• Long-term effects of ethanol are due to acetaldehyde and free-radical production, which leads to fatty liver, hepatitis, and liver cirrhosis.

Formation and Degradation of Glycogen

• Glycogen is the storage form of glucose, composed of glucosyl units linked by α-1,4 glycosidic bonds with α-1,6 branches occurring about every 8 to 10 glucosyl units.
• Glycogen synthesis requires energy.
• Glycogen synthase transfers a glucosyl residue from the activated intermediate UDPglucose to the ends of existing glycogen chains during glycogen synthesis. The branching enzyme creates α-1,6 linkages in the glycogen chain.
• Glycogenolysis is the degradation of glycogen. Glycogen phosphorylase catalyzes a phosphorolysis reaction, utilizing exogenous inorganic phosphate to break α-1,4 linkages at the ends of glycogen chains, releasing glucose 1-phosphate. The debranching enzyme hydrolyzes the α-1,6 linkages in glycogen, releasing free glucose.
• Liver glycogen supplies blood glucose.
• Glycogen synthesis and degradation are regulated in the liver by hormonal changes which signify the need for or excess of blood glucose.
• Lack of dietary glucose, signaled by a decrease of the insulin/glucagon ratio, activates liver glycogenolysis and inhibits glycogen synthesis. Epinephrine also activates liver glycogenolysis.
• Glucagon and epinephrine release lead to phosphorylation of glycogen synthase (inactivating it) and glycogen phosphorylase (activating it).
• Glycogenolysis in muscle supplies glucose 6-phosphate for adenosine triphosphate synthesis in the glycolytic pathway.
• Muscle glycogen phosphorylase is allosterically activated by AMP, as well as by phosphorylation.
• Increases in sarcoplasmic Ca²⁺ stimulate phosphorylation of muscle glycogen phosphorylase.

Digestion, Absorption, and Transport of Carbohydrates

• The major carbohydrates in the American diet are starch, lactose, and sucrose.
• Starch is a polysaccharide composed of many glucose units linked together through α-1,4- and α-1,6-glycosidic bonds (see Fig.)

  

  N- and O-glycosidic bonds. ATP contains a β, N-glycosidic bond. Lactose contains an O-glycosidic β(1→4) bond. Starch contains α-1,4 and α-1,6 O-glycosidic bonds.

• Lactose is a disaccharide composed of glucose and galactose.
• Sucrose is a disaccharide composed of glucose and fructose.
• Digestion converts all dietary carbohydrates to their respective monosaccharides.
• Amylase digests starch; it is found in the saliva and pancreas, which releases it into the small intestine.
• Intestinal epithelial cells contain disaccharidases, which cleave lactose, sucrose, and digestion products of starch into monosaccharides.
• Dietary fiber is composed of polysaccharides that cannot be digested by human enzymes.
• Monosaccharides are transported into the absorptive intestinal epithelial cells via active transport systems.
• Monosaccharides released into the blood via the intestinal epithelial cells are recovered by tissues that utilize facilitative transporters.

Fuel Metabolism by Insulin, Glucagon, and Other Hormones

• Glucose homeostasis is the maintenance of constant blood glucose levels.
• Insulin and glucagon are the two major hormones that regulate fuel mobilization and storage. They maintain blood glucose levels near 80 to 100 mg/dL despite varying carbohydrate intake during the day.
• If dietary intake of all fuels is in excess of immediate need, the excess fuel is stored as either glycogen or fat. Conversely, appropriate stored fuels are mobilized when demand requires.
• Insulin is released in response to carbohydrate ingestion and promotes glucose utilization as a fuel and glucose storage as fat and glycogen. Insulin secretion is regulated principally by blood glucose levels.
• Glucagon promotes glucose production via glycogenolysis (glycogen degradation) and gluconeogenesis (glucose synthesis from amino acids and other noncarbohydrate precursors).
• Glucagon release is regulated principally through suppression by glucose and by insulin. Glucagon levels decrease in response to a carbohydrate meal and increase during fasting. Increased levels of glucagon relative to insulin stimulate the release of fatty acids from adipose tissue.
• Glucagon acts by binding to a receptor on the cell surface, which stimulates the synthesis of the intracellular second messenger cAMP.
• cAMP activates protein kinase A, which phosphorylates key regulatory enzymes, activating some and inhibiting others.
• Insulin acts via a receptor tyrosine kinase and leads to the dephosphorylation of the key enzymes phosphorylated in response to glucagon.

Oxidation of Fatty Acids and Ketone Bodies

• Fatty acids are a major fuel for humans.
• During overnight fasting, fatty acids become the major fuel for cardiac muscle, skeletal muscle, and liver.
• The nervous system has a limited ability to directly use fatty acids as fuel. The liver converts fatty acids to ketone bodies, which can be used by the nervous system as a fuel during prolonged periods of fasting.
• Fatty acids are released from adipose tissue triacylglycerols under appropriate hormonal stimulation.
• In cells, fatty acids are activated to fatty acyl CoA derivatives by acyl CoA synthetases.
• Acyl CoAs are transported into the mitochondria for oxidation via carnitine.
• ATP is generated from fatty acids by the pathway of β-oxidation.
• In β-oxidation, the fatty acyl group is sequentially oxidized to yield FAD(2H), NADH, and acetyl CoA.
• Unsaturated and odd-chain-length fatty acids require additional reactions for their metabolism.
• β-oxidation is regulated by the levels of FAD(2H), NADH and acetyl CoA.
• The entry of fatty acids into mitochondria is regulated by malonyl-CoA levels.
• Alternative pathways for very long chain and branched-chain fatty acid oxidation occur within peroxisomes.

Generation of ATP from Glucose: Glycolysis

• Glycolysis is the pathway in which glucose is oxidized and cleaved to form pyruvate.
• The enzymes of glycolysis are in the cytosol.
• Glucose is the major sugar in our diet; all cells can utilize glucose for energy.
• Glycolysis generates two molecules of ATP through substrate-level phosphorylation and two molecules of NADH.
• The cytosolic NADH generated via glycolysis transfers its reducing equivalents to mitochondrial NAD⁺ via shuttle systems across the inner mitochondrial membrane.
• The pyruvate generated during glycolysis can enter the mitochondria and be oxidized completely to CO₂ by pyruvate dehydrogenase and the TCA cycle.
• Anaerobic glycolysis will generate energy in cells with a limited supply of oxygen or few mitochondria.
• Under anaerobic conditions, pyruvate is reduced to lactate by NADH, thereby regenerating the NAD⁺ required for glycolysis to continue.
• Glycolysis is regulated to ensure that ATP homeostasis is maintained.

Oxidative Phosphorylation, Mitochondrial Function, and Oxygen Radicals

• The reduced cofactors generated during fuel oxidation donate their electrons to the mitochondrial electron transport chain.
• The electron transport chain transfers the electrons to O₂, which is reduced to water.
• As electrons travel through the electron transport chain, protons are transferred from the mitochondrial matrix to the cytosolic side of the inner mitochondrial membrane.
• The asymmetrical distribution of protons across the inner mitochondrial membrane generates an electrochemical gradient across the membrane.
  The electrochemical gradient consists of a change in pH (ΔpH) across the membrane, and a difference in charge (ΔΦ) across the membrane.
• Proton entry into the mitochondrial matrix is energetically favorable and drives the synthesis of ATP via the ATP synthase.
• Respiration (O₂ consumption) is normally coupled to ATP synthesis; if one process is inhibited, the other is also inhibited.
• Uncouplers allow respiration to continue in the absence of ATP synthesis, as the energy inherent in the proton gradient is released as heat.
• Through a number of enzymatic and nonenzymatic processes O₂ can accept a single electron to form reactive oxygen species.
• The major reactive oxygen species are the radicals superoxide and hydroxyl radical and the nonradical hydrogen peroxide.
• Reactive oxygen species cause damage to lipids, proteins, and DNA within cells.
• Cellular defense mechanisms exist to protect against inadvertent radical formation.

Tricarboxylic Acid Cycle

• The TCA cycle accounts for over two-thirds of the ATP generated from fuel oxidation.
• Acetyl CoA, generated from fuel oxidation, is the substrate for the TCA cycle.
• Acetyl CoA, when oxidized via the cycle, generates CO₂, reduced electron carriers, and guanosine triphosphate.
• The reduced electron carriers (NADH, FAD[2H]) donate electrons to O₂ via the electron transport chain, which leads to ATP generation from oxidative phosphorylation.
• The cycle requires a number of cofactors to function properly, some of which are derived from vitamins. These include thiamine pyrophosphate (derived from vitamin B₁), FAD (derived from vitamin B₂) and coenzyme A (derived from pantothenic acid).
• Intermediates of the TCA cycle are used for many biosynthetic reactions and are replaced by anaplerotic (refilling) reactions within the cell.
• The cycle is carefully regulated within the mitochondria by energy and the levels of reduced electron carriers.
• Impaired functioning of the TCA cycle leads to an inability to generate ATP from fuel oxidation and an accumulation of TCA cycle precursors.

Cellular Bioenergetics: ATP and O2

• Bioenergetics refers to cellular energy transformations.
• The high-energy phosphate bonds of ATP are a cell's primary source of energy.
• ATP is generated through cellular respiration, the oxidation of fuels to carbon dioxide and water.
• The electrons captured from fuel oxidation regenerate ATP via the process of oxidative phosphorylation.
• The energy available from ATP hydrolysis can be used for
  • Mechanical work (muscle contraction)
  • Transport work (establishment of ion gradients across membranes)
  • Biochemical work (energy-requiring chemical reactions)
• Energy released from fuel oxidation that is not used for work is transformed into and released as heat.
• Fuel oxidation is regulated to maintain ATP homeostasis.
• ΔG⁰′ is the change in Gibbs free energy at pH 7.0 under standard conditions.
• Fuel oxidation has a negative ΔG⁰′; the products formed have a lower chemical energy than the reactants (an exergonic reaction pathway).
• ATP synthesis has a positive ΔG⁰′ and is endergonic; the reaction requires energy.
• Metabolic pathways have an overall negative ΔG⁰′.

Use of Recombinant DNA Techniques in Medicine

• Techniques for isolating and amplifying genes and studying and manipulating DNA sequences are currently being used in the diagnosis, prevention, and treatment of disease.
• These techniques require an understanding of the following tools and processes:
  • Restriction enzymes
  • Cloning vectors
  • Polymerase chain reaction
  • Gel electrophoresis
  • Nucleic acid hybridization
  • Expression vectors
• Recombinant DNA molecules produced by these techniques can be used as diagnostic probes, in gene therapy, or for the large-scale production of proteins for the treatment of disease.
• Identified genetic polymorphisms, inherited differences in DNA base sequences between individuals, can be utilized for both diagnosis of disease and the generation of an individual's molecular fingerprint.

Regulation of Gene Expression

• Prokaryotic gene expression is primarily regulated at the level of initiation of gene transcription. In general, there is one protein per gene.
  • Sets of genes encoding proteins with related functions are organized into operons.
  • Each operon is under the control of a single promoter.
  • Repressors bind to the promoter to inhibit RNA polymerase binding.
  • Activators facilitate RNA polymerase binding to the repressor.
• Eukaryotic gene regulation occurs at several levels.
  • At the DNA structural level chromatin must be remodeled to allow access for RNA polymerase.
  • Transcription is regulated by transcription factors, which either enhance or restrict RNA polymerase access to the promoter.
  • RNA processing (including alternative splicing), transport from the nucleus to the cytoplasm, and translation are also regulated in eukaryotes.

Translation: Synthesis of Proteins

• Translation is the process of translating the sequence of nucleotides in mRNA to an amino acid sequence of a protein.
• Translation proceeds from the amino to carboxy terminal, reading the mRNA in the 5′ to 3′ direction.
• Protein synthesis occurs on ribosomes.
• The mRNA is read in codons, sets of three nucleotides that specify individual amino acids.
• AUG, which specifies methionine, is the start codon for all protein synthesis.
• Specific stop codons (UAG, UGA, and UAA) signal when the translation of the mRNA is to end.
• Amino acids are covalently linked to tRNA by the enzyme aminoacyl-tRNA synthetase, creating charged tRNA.
• Charged tRNAs base-pair with the codon via the anticodon region of the tRNA.
• Protein synthesis is divided into three stages: initiation, elongation, and termination.
• Multiprotein factors are required for each stage of protein synthesis.
• Proteins fold as they are synthesized.
• Specific amino acid side chains may be modified after translation, in a process known as posttranslational modification.
• Mechanisms within cells specifically target newly synthesized proteins to different compartments in the cell.

Transcription: Synthesis of RNA

• Transcription is the synthesis of RNA from a DNA template.
• The enzyme RNA polymerase transcribes genes into a single-stranded RNA.
• The RNA produced is complementary to one of the strands of DNA, which is known as the template strand. The other DNA strand is the coding, or sense strand.
• Bacteria contain a single RNA polymerase; eukaryotic cells utilize three different RNA polymerases.
• The DNA template is copied in the 3' to 5' direction and the RNA transcript is synthesized in the 5' to 3' direction.
• In contrast to DNA polymerases, RNA polymerases do not require a primer to initiate transcription, nor do they contain error-checking capabilities.
• Promoter regions, specific sequences in DNA, determine where on the DNA template RNA polymerase binds to initiate transcription.
• Transcription initiation requires a number of protein factors to allow for efficient RNA polymerase binding to the promoter.
• Other DNA sequences, such as promoter-proximal elements and enhancers, affect the rate of transcription initiation through the interactions of DNA-binding proteins with RNA polymerase and other initiation factors.
• Eukaryotic genes contain exons and introns. Exons specify the coding region of proteins, whereas introns have no coding function.
• The primary transcript of eukaryotic genes is modified to remove the introns (splicing) before a final, mature mRNA is produced.

Synthesis of DNA

• Replication of the genome requires DNA synthesis.
• During replication, each of the two parental strands of DNA serves as a template for the synthesis of a complementary strand.
• The site at which replication is occurring is called the replication fork.
• Helicases and topoisomerases are required to unwind the DNA helix of the parental strands.
• DNA polymerase is the major enzyme involved in replication.
• DNA polymerase copies each parental template strand in the 3' to 5' direction, producing new strands in a 5' to 3' direction.
• The precursors for replication are deoxyribonucleotide triphosphates.
• As DNA synthesis proceeds in the 5' to 3' direction, one parental strand is synthesized continuously, whereas the other exhibits discontinuous synthesis, creating small fragments which are subsequently joined, because DNA polymerase must synthesize DNA in the 5' to 3' direction.
• DNA polymerase requires a free 3' hydroxyl group of a nucleotide primer in order to replicate DNA. The primer is synthesized by the enzyme primase, which provides an RNA primer.
• The enzyme telomerase synthesizes the replication of the ends of linear chromosomes (telomeres).
• Errors during replication can lead to mutations, so error checking and repair systems function to maintain the integrity of the genome.

Structure of the Nucleic Acids

• The central dogma of molecular biology is that DNA is transcribed to RNA, which is translated to protein.
• Nucleotides, consisting of a nitrogenous base, a five-carbon sugar, and phosphate, are the monomeric unit of the nucleic acids DNA and RNA (see Chapter 3).
• DNA contains the sugar 2′-deoxyribose; RNA contains ribose.
• DNA and RNA contain the purine bases adenine (A) and guanine (G).
• DNA contains the pyrimidine bases cytosine (C) and thymine (T), whereas RNA contains C and uracil (U).
• DNA and RNA are linear sequences of nucleotides linked by phosphodiester bonds between the 3′ sugar of one nucleotide and the 5′ sugar of the next nucleotide.
• Genetic information is encoded by the sequence of the nucleotide bases in DNA.
• DNA is double stranded; one strand runs in the 5′ to 3′ direction, while the other is antiparallel and runs in the 3′ to 5′ direction.
• The two strands of DNA wrap about each other to form a double helix and are held together by hydrogen bonding between bases in each strand.
• A hydrogen bonds to T, while C hydrogen bonds to G.
• Transcription of a gene generates a single-stranded RNA; the three major types of RNA are messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA).
• Eukaryotic mRNA is modified at both the 5′ and 3′ ends. In between it contains a coding region for the synthesis of a protein.
• Codons within the coding region dictate the sequence of amino acids in a protein. Each codon is three nucleotides long.
• rRNA and tRNA are required for protein synthesis.
• rRNA is complexed with proteins to form ribonucleoprotein particles called ribosomes, which bind mRNA and tRNAs during translation.
  The tRNA contains an anticodon which binds to a complementary codon on mRNA, ensuring insertion of the correct amino acid into the protein being synthesized.

Cell Structure and Signaling by Chemical Messengers

• The cell is the basic unit of living organisms.
• Unique features of each cell type define tissue specificity and function.
• Despite the variety of cell types, cells share many common features, which include a plasma membrane and intracellular organelles.
• In eukaryotes, the intracellular organelles consist of lysosomes, the nucleus, ribosomes, the endoplasmic reticulum, the Golgi apparatus, mitochondria, peroxisomes, and the cytoplasm. Some cells may lack one or more of these internal organelles.
• In order to integrate cellular function with the needs of the organism, cells communicate with each other via chemical messengers. Chemical messengers include neurotransmitters (for the nervous system), hormones (for the endocrine system), cytokines (for the immune system), retinoids, eicosanoids, and growth factors.
• Chemical messengers transmit their signals by binding to receptors on target cells. When a messenger binds to a receptor, a signal transduction pathway is activated and generates second messengers within the cell.
• Receptors can be either plasma membrane proteins or intracellular binding G-proteins.
  Intracellular receptors act primarily as transcription factors, which regulate gene expression in response to a signal being released.
• Plasma membrane receptors fall into different classes, such as ion channel receptors, tyrosine kinase receptors, tyrosine kinase–associated receptors, serine-threonine kinase receptors, or G-protein–coupled receptors (GPCR).

Regulation of Enzymes

• Enzyme activity is regulated to reflect the physiological state of the organism.
• The rate of an enzyme-catalyzed reaction is dependent on substrate concentration and can be represented mathematically by the Michaelis-Menten equation.
• Allosteric activators or inhibitors are compounds that bind at sites other than the active catalytic site and regulate the enzyme through conformational changes affecting the catalytic site.
• A number of different mechanisms are available to regulate enzyme activity. These include:
• Feedback inhibition, which often occurs at the first committed step of a metabolic pathway
• Covalent modification of an amino acid residue (or residues) within the protein
• Interactions with modulator proteins, which when bound to the enzyme, alter the conformation of the enzyme, and hence activity
  Altering the primary structure of the protein via proteolysis
• Increasing or decreasing the amount of enzyme available in the cell via alterations in the rate of synthesis of degradation of the enzyme
• Metabolic pathways are frequently regulated at the slowest, or rate-limiting, step of the pathway.

Enzymes as Catalysts

• Enzymes are proteins that act as catalysts—molecules that can accelerate the rate of a reaction.
• Enzymes are specific for various substrates because of the selective nature of the binding sites on the enzyme.
• The catalytic (active) site is the portion of the enzyme molecule at which the reaction occurs.
• Enzymes accelerate reaction rates by decreasing the amount of energy required to reach a high-energy intermediate stage of the reaction, known as the transition state complex. This is referred to as lowering the energy of activation.
• Enzymes utilize functional groups at the active site, provided by coenzymes, metals, or amino acid residues, to perform catalysis.
• Enzymes utilize general acid-base catalysis, formation of covalent intermediates, and transition state stabilization as various mechanisms to accelerate reaction rates.
• Many drugs and toxins act by inhibiting enzymes.
• Enzymes can be regulated to control reaction rates through a variety of mechanisms.

Structural Function Relationships in Proteins

• There are four levels of protein structure:
• The primary structure (linear sequence of amino acids within the protein)
• The secondary structure (a regular, repeating pattern of hydrogen bonds stabilizing a particular structure)
• The tertiary structure (the folding of the secondary structure elements into a three-dimensional conformation)
• The quaternary structure (the association of subunits within a protein)
• The primary structure of a protein determines the way a protein folds into a unique three-dimensional structure, called its native conformation.
• When globular proteins fold, the tertiary structure generally forms a densely packed hydrophobic core with polar amino acid side chains on the outside, facing the aqueous environment.
• The tertiary structure of a protein consists of structural domains, which may be similar between different proteins and perform similar functions for the different proteins.
• Certain structural domains are binding sites for specific molecules, called a ligand, or for other proteins.
• The affinity of a binding site for its ligand is quantitatively characterized by an association or affinity constant, K_a (or dissociation constant, K_d).
• Protein denaturation is the loss of tertiary (and/or secondary) structure within a protein, which can be caused by heat, acid, or other agents that interfere with hydrogen bonding and usually causes a decrease in solubility (precipitation).

Amino Acids and Proteins

• A protein's unique characteristics, including its three-dimensional folded structure, are dictated by its linear sequence of amino acids, termed its primary structure.
• The primary structures of all of the diverse human proteins are synthesized from 20 amino acids arranged in a linear sequence determined by the genetic code.
• Each three-base (nucleotide) sequence within the coding region of a gene (the genetic code) specifies which amino acid should be present in a protein. The genetic code is discussed further in Chapter 12.
• All amino acids contain a central α-carbon, joined to a carboxylic acid group, an amino group, a hydrogen, and a side chain, which varies between the 20 different amino acids.
• At physiological pH the amino acids are zwitterions; the amino group is positively charged, and the carboxylate is negatively charged.
• In proteins, amino acids are joined into linear polymers called polypeptide chains via peptide bonds, which are formed between the carboxylic acid of one amino acid and the amino group of the next amino acid.
• Amino acid side chains can be classified either by polarity (charged, nonpolar hydrophobic, or uncharged polar) or structural features (aliphatic, cyclic, or aromatic).
• Depending on their side chain characteristics, certain amino acids cluster together to exclude water (hydrophobic effect), whereas others participate in hydrogen bonding. Cysteine can form disulfide bonds, whereas charged amino acids can form ionic bonds.
• Amino acids in proteins can be modified by phosphorylation, carboxylation, or other reactions after the protein is synthesized (posttranslational modifications).
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• Alterations in the genetic code may lead to mutations in the protein's primary structure, which can affect the protein's function.
• Proteins with the same function but different primary structure (isoforms and isozymes) can exist in different tissues or during different phases of development.

The Major Compounds of the Body

• Carbohydrates, commonly known as sugars, can be classified by a number of criteria:
• Type of carbonyl group (aldo or keto sugars)
• Number of carbons (pentoses [5 carbons], hexoses [6 carbons])
• Positions of hydroxyl groups on asymmetrical carbon atoms (d or l configuration, stereoisomers, epimers)
• Substituents (amino sugars)
• Number of monosaccharides joined through glycosidic bonds (disaccharides, oligosaccharides, polysaccharides)
• Lipids are structurally diverse compounds that are not very soluble in water (i.e., they are hydrophobic).
• The major lipids are fatty acids.
• Triacylglycerols (triglycerides) consist of three fatty acids esterified to the carbohydrate glycerol.
• Phosphoacylglycerols (phosphoglycerides or phospholipids) are similar to triacylglycerol but contain a phosphate in place of a fatty acid.
• Sphingolipids are built upon sphingosine.
• Cholesterol is a component of membranes and a precursor for molecules which contain the steroid nucleus, such as bile salts and steroid hormones.
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• Nitrogen is found in a variety of compounds in addition to amino sugars.
• Amino acids and heterocyclic rings contain nitrogens, which carry a positive charge at neutral pH.
• Amino acids contain a carboxyl group, an amino group, and a side chain attached to a central carbon.
• Proteins consist of a linear chain of amino acids.
• Purines, pyrimidines, and pyridines have heterocyclic nitrogen-containing ring structures.
• Nucleosides consist of a heterocyclic ring attached to a sugar.
• A nucleoside plus phosphate is a nucleotide.
• Glycoproteins and proteoglycans have sugars attached to protein components.