Biochemistry_Lippinco

Biochemistry_Lippinco. The nonoxidative reactions of the pentose phosphate pathway occur in all cell types synthesizing nucleotides and nucleic acids. These reactions catalyze the interconversion of sugars containing three to seven carbons (see Fig. 13.2). These reversible reactions permit ribulose 5-phosphate (produced by the oxidative portion of the pathway) to be converted either to ribose 5-phosphate (needed for nucleotide synthesis; see p. 293) or to intermediates of glycolysis (that is, fructose 6-phosphate and glyceraldehyde 3-phosphate). For example, many cells that carry out reductive biosynthetic reactions have a greater need for NADPH than for ribose 5-phosphate. In this case, transketolase (which transfers two-carbon units in a thiamine pyrophosphate [TPP]-requiring reaction) and transaldolase (which transfers three-carbon units) convert the ribulose 5phosphate produced as an end product of the oxidative phase to glyceraldehyde 3-phosphate and fructose 6-phosphate, which are glycolytic


Biochemistry_Lippinco

Biochemistry_Lippinco. (which transfers three-carbon units) convert the ribulose 5phosphate produced as an end product of the oxidative phase to glyceraldehyde 3-phosphate and fructose 6-phosphate, which are glycolytic intermediates. In contrast, when the demand for ribose for nucleotides and nucleic acids is greater than the need for NADPH, the nonoxidative reactions can provide the ribose 5phosphate from glyceraldehyde 3-phosphate and fructose 6-phosphate in the absence of the oxidative steps (Fig. 13.3).


Biochemistry_Lippinco

Biochemistry_Lippinco. In addition to transketolase, TPP is required by the multienzyme complexes pyruvate dehydrogenase (see p. 110), α-ketoglutarate dehydrogenase of the tricarboxylic acid cycle (see p. 112), and branched-chain α-keto acid dehydrogenase of branched-chain amino acid catabolism (see p. 266). IV. NADPH USES The coenzyme NADPH differs from nicotinamide adenine dinucleotide (NADH) only by the presence of a phosphate group on one of the ribose units (Fig. 13.4). This seemingly small change in structure allows NADPH to interact with NADPH-specific enzymes that have unique roles in the cell. For example, in the cytosol of hepatocytes, the steady-state NADP+/NADPH ratio is ~0.1, which favors the use of NADPH in reductive biosynthetic reactions. This contrasts with the high NAD+/NADH ratio (~1,000), which favors an oxidative role for NAD+. This section summarizes some important NADPH-specific functions in reductive biosynthesis and detoxification reactions. A. Reductive biosynthesis


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Reductive biosynthesis Like NADH, NADPH can be thought of as a high-energy molecule. However, the electrons of NADPH are used for reductive biosynthesis, rather than for transfer to the electron transport chain as is seen with NADH (see p. 74). Thus, in the metabolic transformations of the pentose phosphate pathway, part of the energy of glucose 6-phosphate is conserved in NADPH, a molecule with a negative reduction potential (see p. 76), that, therefore, can be used in reactions requiring an electron donor, such as fatty acid (see p. 186), cholesterol (see p. 221), and steroid hormone (see p. 237) synthesis. B. Hydrogen peroxide reduction


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Hydrogen peroxide reduction Hydrogen peroxide (H2O2) is one of a family of reactive oxygen species (ROS) that are formed from the partial reduction of molecular oxygen ([O2], Fig. 13.5A). These compounds are formed continuously as byproducts of aerobic metabolism, through reactions with drugs and environmental toxins, or when the level of antioxidants is diminished, all creating the condition of oxidative stress. These highly reactive oxygen intermediates can cause serious chemical damage to DNA, proteins, and unsaturated lipids and can lead to cell death. ROS have been implicated in a number of pathologic processes, including reperfusion injury, cancer, inflammatory disease, and aging. The cell has several protective mechanisms that minimize the toxic potential of these compounds. [Note: ROS can also be generated in the killing of microbes by white blood cells (WBC; see D. below).]


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Actions of antioxidant enzymes. G-SH = reduced glutathione; G-S-S-G = oxidized glutathione. [Note: See Fig. 13.6B for the regeneration of G-SH.] 1. Enzymes that catalyze antioxidant reactions Reduced glutathione (GSH), a tripeptide-thiol (γ-glutamylcysteinylglycine) present in most cells, can chemically detoxify H2O2 (Fig. 13.5B). This reaction, catalyzed by the selenoprotein (see p. 407) glutathione peroxidase, forms oxidized glutathione (G-S-S-G), which no longer has protective properties. The cell regenerates G-SH in a reaction catalyzed by glutathione reductase, using NADPH as a source of reducing equivalents. Thus, NADPH indirectly provides electrons for the reduction of H2O2 (Fig. 13.6). Additional enzymes, such as superoxide dismutase and catalase, catalyze the conversion of other ROS to harmless products (see Fig. 13.5B). As a group, these enzymes serve as a defense system to guard against the toxic effects of ROS. glutathione.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Antioxidant chemicals A number of intracellular reducing agents, such as ascorbate (see p. 381), vitamin E (see p. 395), and β-carotene (see p. 386), are able to reduce and, thereby, detoxify ROS in the laboratory. Consumption of foods rich in these antioxidant compounds has been correlated with a reduced risk for certain types of cancers as well as decreased frequency of certain other chronic health problems. Therefore, it is tempting to speculate that the effects of these compounds are, in part, an expression of their ability to quench the toxic effect of ROS. However, clinical trials with antioxidants as dietary supplements have failed to show clear beneficial effects. In the case of dietary supplementation with β-carotene, the rate of lung cancer in smokers increased rather than decreased. Thus, the health-promoting effects of dietary fruits and vegetables likely reflect a complex interaction among many naturally occurring compounds, which has not been duplicated by consumption


Biochemistry_Lippinco

Biochemistry_Lippinco. Thus, the health-promoting effects of dietary fruits and vegetables likely reflect a complex interaction among many naturally occurring compounds, which has not been duplicated by consumption of isolated antioxidant compounds.


Biochemistry_Lippinco

C. Cytochrome P450 monooxygenase system

Biochemistry_Lippinco. C. Cytochrome P450 monooxygenase system


Biochemistry_Lippinco

Biochemistry_Lippinco. Monooxygenases (mixed-function oxidases) incorporate one atom from O2 into a substrate (creating a hydroxyl group), with the other atom being reduced to water (H2O). In the cytochrome P450 (CYP) monooxygenase system, NADPH provides the reducing equivalents required by this series of reactions (Fig. 13.7). This system performs different functions in two separate locations in cells. The overall reaction catalyzed by a CYP enzyme is where R may be a steroid, drug, or other chemical. [Note: CYP enzymes are actually a superfamily of related, heme-containing monooxygenases that participate in a broad variety of reactions. The P450 in the name reflects the absorbance at 450 nm by the protein.] 1. Mitochondrial system An important function of the CYP monooxygenase system found associated with the inner mitochondrial membrane is the biosynthesis of steroid hormones. In steroidogenic tissues, such as the placenta, ovaries, testes, and adrenal cortex, it is used to hydroxylate intermediates in


Biochemistry_Lippinco

Biochemistry_Lippinco. inner mitochondrial membrane is the biosynthesis of steroid hormones. In steroidogenic tissues, such as the placenta, ovaries, testes, and adrenal cortex, it is used to hydroxylate intermediates in the conversion of cholesterol to steroid hormones, a process that makes these hydrophobic compounds more water soluble (see p. 237). The liver uses this same system in bile acid synthesis (see p.


Biochemistry_Lippinco

Biochemistry_Lippinco. 224) and the hydroxylation of cholecalciferol to 25hydroxycholecalciferol ([vitamin D3] see p. 390), and the kidney uses it to hydroxylate vitamin D3 to its biologically active 1,25-dihydroxylated form.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Microsomal system The microsomal CYP monooxygenase system found associated with the membrane of the smooth endoplasmic reticulum (particularly in the liver) functions primarily in the detoxification of foreign compounds (xenobiotics). These include numerous drugs and such varied pollutants as petroleum products and pesticides. CYP enzymes of the microsomal system (for example, CYP3A4) can be used to hydroxylate these toxins (phase I). The purpose of these modifications is two-fold. First, it may itself activate or inactivate a drug and second, make a toxic compound more soluble, thereby facilitating its excretion in the urine or feces. Frequently, however, the new hydroxyl group will serve as a site for conjugation with a polar molecule, such as glucuronic acid (see p. 161), which will significantly increase the compound’s solubility (phase II). [Note: Polymorphisms (see p. 491) in the genes for CYP enzymes can lead to differences in drug metabolism.]


Biochemistry_Lippinco

Biochemistry_Lippinco. D. White blood cell phagocytosis and microbe killing Phagocytosis is the ingestion by receptor-mediated endocytosis of microorganisms, foreign particles, and cellular debris by WBC (leukocytes) such as neutrophils and macrophages (monocytes). It is an important defense mechanism, particularly in bacterial infections. Neutrophils and monocytes are armed with both oxygen-independent and oxygen-dependent mechanisms for killing bacteria. 1. Oxygen-independent Oxygen-independent mechanisms use pH changes in phagolysosomes and lysosomal enzymes to destroy pathogens. 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. Oxygen-dependent Oxygen-dependent mechanisms include the enzymes NADPH oxidase and myeloperoxidase (MPO) that work together in killing bacteria (Fig. 13.8). Overall, the MPO system is the most potent of the bactericidal mechanisms. An invading bacterium is recognized by the immune system and attacked by antibodies that bind it to a receptor on a phagocytic cell. After internalization of the microorganism has occurred, NADPH oxidase, located in the leukocyte cell membrane, activated and reduces O2 from the surrounding tissue to superoxide ( ), a free radical ROS, as NADPH is oxidized. The rapid consumption of O2 that accompanies formation of is referred to as the respiratory burst. [Note: Active NADPH oxidase is a membrane-associated complex containing a flavocytochrome plus additional peptides that translocate from the cytoplasm upon activation of the leukocyte. Electrons move from NADPH to O2 via flavin adenine nucleotide (FAD) and heme, . Rare genetic deficiencies in NADPH oxidase


Biochemistry_Lippinco

Biochemistry_Lippinco. that translocate from the cytoplasm upon activation of the leukocyte. Electrons move from NADPH to O2 via flavin adenine nucleotide (FAD) and heme, . Rare genetic deficiencies in NADPH oxidase cause chronic granulomatous disease (CGD) characterized by severe, persistent infections and the formation of granulomas (nodular areas of inflammation) that sequester the bacteria that were not destroyed.] Next, is converted to H2O2 (also a ROS), either spontaneously or catalyzed by superoxide dismutase. In the presence of MPO, a heme-containing lysosomal enzyme present within the phagolysosome, peroxide plus chloride ions are converted to hypochlorous acid ([HOCl] the major component of household bleach), which kills the bacteria. The peroxide can also be partially reduced to the hydroxyl radical (OH•), a


Biochemistry_Lippinco

Biochemistry_Lippinco. ROS, or be fully reduced to H2O by catalase or glutathione peroxidase. [Note: Deficiencies in MPO do not confer increased susceptibility to infection because peroxide from NADPH oxidase is bactericidal.] G; NADP(H) = nicotinamide adenine dinucleotide phosphate; = superoxide; H2O2 = hydrogen peroxide; HOCl = hypochlorous acid; OH• = hydroxyl radical. E. Nitric oxide synthesis


Biochemistry_Lippinco

Biochemistry_Lippinco. Nitric oxide (NO) is recognized as a mediator in a broad array of biologic systems. NO is the endothelium-derived relaxing factor that causes vasodilation by relaxing vascular smooth muscle. It also acts as a neurotransmitter, prevents platelet aggregation, and plays an essential role in macrophage function. It has a very short half-life in tissues (3–10 seconds) because it reacts with O2 and and is converted into nitrates and nitrites including peroxynitrite (O=NOO−), a reactive nitrogen species (RNS). [Note: NO is a free radical gas that is often confused with nitrous oxide (N2O), the “laughing gas” that is used as an anesthetic and is chemically stable.] 1. Nitric oxide synthase Arginine, O2, and NADPH are substrates for cytosolic NO synthase ([NOS], Fig. 13.9). Flavin mononucleotide (FMN), FAD, heme, and tetrahydrobiopterin (see p. 268) are coenzymes, and NO and citrulline are products of the reaction. Three NOS isozymes, each the product of a different gene, have been identified.


Biochemistry_Lippinco

Biochemistry_Lippinco. FAD, heme, and tetrahydrobiopterin (see p. 268) are coenzymes, and NO and citrulline are products of the reaction. Three NOS isozymes, each the product of a different gene, have been identified. Two are constitutive (synthesized at a constant rate), calcium (Ca2+)–calmodulin (CaM)-dependent enzymes (see p. 133). They are found primarily in endothelium (eNOS) and neural tissue (nNOS) and constantly produce very low levels of NO for vasodilation and neurotransmission. An inducible, Ca2+-independent enzyme (iNOS) can be expressed in many cells, including macrophages and neutrophils, as an early defense against pathogens. The specific inducers for iNOS vary with cell type and include proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), and bacterial endotoxins such as lipopolysaccharide (LPS). These compounds promote synthesis of iNOS, which can result in large amounts of NO being produced over hours or even days.


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Biochemistry_Lippinco. 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. Nitric oxide and vascular endothelium NO is an important mediator in the control of vascular smooth muscle tone. NO is synthesized by eNOS in endothelial cells and diffuses to vascular smooth muscle, where it activates the cytosolic form of guanylyl cyclase (or, guanylate cyclase) to form cyclic guanosine monophosphate (cGMP). [Note: This reaction is analogous to the formation of cyclic adenosine monophosphate (cAMP) by adenylyl cyclase (see p. 95).] The resultant rise in cGMP causes activation of protein kinase G, which phosphorylates Ca2+ channels, causing decreased entry of Ca2+ into smooth muscle cells. This decreases the Ca2+–CaM activation of myosin light-chain kinase, thereby decreasing smooth muscle contraction and favoring relaxation. Vasodilator nitrates, such as nitroglycerin, are metabolized to NO, which causes relaxation of vascular smooth muscle and, therefore, lowers blood pressure. Thus, NO can be envisioned as an endogenous nitrovasodilator. [Note: Under hypoxic


Biochemistry_Lippinco

Biochemistry_Lippinco. are metabolized to NO, which causes relaxation of vascular smooth muscle and, therefore, lowers blood pressure. Thus, NO can be envisioned as an endogenous nitrovasodilator. [Note: Under hypoxic conditions, nitrite (NO2−) can be reduced to NO, which binds to deoxyhemoglobin. The NO is released into the blood, causing vasodilation and increasing blood flow.] 3.


Biochemistry_Lippinco

Biochemistry_Lippinco. Nitric oxide and macrophage bactericidal activity In macrophages, iNOS activity is normally low, but synthesis of the enzyme is significantly stimulated by bacterial LPS and by release of IFN-γ response to infection. Activated macrophages form radicals that combine with NO to form intermediates that decompose, producing the highly bactericidal OH• radical. 4. Additional functions NO is a potent inhibitor of platelet adhesion and aggregation (by activating the cGMP pathway). It is also characterized as a neurotransmitter in the central and peripheral nervous systems. V. G6PD DEFICIENCY


Biochemistry_Lippinco

Biochemistry_Lippinco. G6PD deficiency is a hereditary condition characterized by hemolytic anemia caused by the inability to detoxify oxidizing agents. G6PD deficiency is the most common disease-producing enzyme abnormality in humans, affecting >400 million individuals worldwide. This deficiency has the highest prevalence in the Middle East, tropical Africa and Asia, and parts of the Mediterranean. G6PD deficiency is X linked and is, in fact, a family of deficiencies caused by a number of different mutations in the gene encoding G6PD. Only some of the resulting protein variants cause clinical symptoms. [Note: In addition to hemolytic anemia, a clinical manifestation of G6PD deficiency is neonatal jaundice appearing 1–4 days after birth. The jaundice, which may be severe, typically results from increased production of unconjugated bilirubin (see p. 285).] The life span of individuals with a severe form of G6PD deficiency may be somewhat shortened as a result of complications arising from chronic hemolysis.


Biochemistry_Lippinco

Biochemistry_Lippinco. of unconjugated bilirubin (see p. 285).] The life span of individuals with a severe form of G6PD deficiency may be somewhat shortened as a result of complications arising from chronic hemolysis. This negative effect of G6PD deficiency has been balanced in evolution by an advantage in survival, an increased resistance to Plasmodium falciparum malaria. [Note: Sickle cell trait and the thalassemias also confer resistance to malaria.]


Biochemistry_Lippinco

A. G6PD role in red blood cells

Biochemistry_Lippinco. A. G6PD role in red blood cells


Biochemistry_Lippinco

Biochemistry_Lippinco. Diminished G6PD activity impairs the ability of the cell to form the NADPH that is essential for the maintenance of the G-SH pool. This results in a decrease in the detoxification of free radicals and peroxides formed within the cell (Fig. 13.10). G-SH also helps maintain the reduced states of sulfhydryl groups in proteins, including hemoglobin. Oxidation of those sulfhydryl groups leads to the formation of denatured proteins that form insoluble masses (called Heinz bodies) that attach to RBC membranes (Fig. 13.11). Additional oxidation of membrane proteins causes RBC to be rigid (less deformable), and they are removed from the circulation by macrophages in the spleen and liver. Although G6PD deficiency occurs in all cells of the affected individual, it is most severe in RBC, where the pentose phosphate pathway provides the only means of generating NADPH. Additionally, the RBC has no nucleus or ribosomes and cannot renew its supply of the enzyme. Thus, RBC are particularly vulnerable


Biochemistry_Lippinco

Biochemistry_Lippinco. phosphate pathway provides the only means of generating NADPH. Additionally, the RBC has no nucleus or ribosomes and cannot renew its supply of the enzyme. Thus, RBC are particularly vulnerable to enzyme variants with diminished stability. [Note: Other tissues have an alternative source of NADPH (NADP+-dependent malate dehydrogenase [malic enzyme]; see p. 186) that can keep G-SH reduced.] pentose phosphate pathway.


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Precipitating factors in G6PD deficiency Most individuals who have inherited one of the G6PD mutations do not show clinical manifestations (that is, they are asymptomatic). However, some patients with G6PD deficiency develop hemolytic anemia if they are treated with an oxidant drug, ingest fava beans, or contract a severe infection. 1. Oxidant drugs Commonly used drugs that produce hemolytic anemia in patients with G6PD deficiency are best remembered from the mnemonic AAA: antibiotics (for example, sulfamethoxazole and chloramphenicol), antimalarials (for example, primaquine but not chloroquine or quinine), and antipyretics (for example, acetanilide but not acetaminophen). 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Favism Some forms of G6PD deficiency, for example, the Mediterranean variant, are particularly susceptible to the hemolytic effect of the fava (broad) bean, a dietary staple in the Mediterranean region. Favism, the hemolytic effect of ingesting fava beans, is not observed in all individuals with G6PD deficiency, but all patients with favism have G6PD deficiency. 3. Infection Infection is the most common precipitating factor of hemolysis in G6PD deficiency. The inflammatory response to infection results in the generation of free radicals in macrophages. The radicals can diffuse into the RBC and cause oxidative damage. C. Variant G6PD properties


Biochemistry_Lippinco

Biochemistry_Lippinco. Almost all G6PD variants are caused by point mutations (see p. 449) in the gene for G6PD. Some mutations do not affect enzymic activity. However, other mutations result in decreased catalytic activity, decreased stability, or an alteration of binding affinity for NADP+ or glucose 6-phosphate. [Note: Active G6PD exists as a homodimer or tetramer. Mutations at the interface between subunits can affect stability.] The severity of the disease usually correlates with the amount of residual enzyme activity in the patient’s RBC. For example, variants can be classified as shown in Figure 13.12. G6PD A– is the prototype of the moderate (class III) form of the disease. The RBC contain an unstable but kinetically normal G6PD, with most of the enzyme activity present in the reticulocytes and younger RBC (Fig. 13.13). Therefore, the oldest RBC have the lowest level of enzyme activity and are preferentially removed in a hemolytic episode. Because hemolysis does not affect younger cells, the


Biochemistry_Lippinco

Biochemistry_Lippinco. RBC (Fig. 13.13). Therefore, the oldest RBC have the lowest level of enzyme activity and are preferentially removed in a hemolytic episode. Because hemolysis does not affect younger cells, the episodes are self-limiting. G6PD Mediterranean is the prototype of a more severe (class II) deficiency. Class I mutations (rare) are the most severe and are associated with chronic nonspherocytic hemolytic anemia, which occurs even in the absence of oxidative stress.


Biochemistry_Lippinco

Biochemistry_Lippinco. D. G6PD molecular biology The cloning of the gene for G6PD and the sequencing of its DNA (see Chapter 34) have permitted the identification of mutations that cause G6PD deficiency. More than 400 G6PD variants have been identified, a finding that explains the numerous biochemical and clinical phenotypes that have been described. Most mutations that result in enzymic deficiency are missense mutations (see p. 449) in the coding region. Both G6PD A− and G6PD Mediterranean represent mutant enzymes that differ from the respective normal variants by a single amino acid. Large deletions or frameshift mutations have not been identified, suggesting that complete absence of G6PD activity is likely lethal. VI. CHAPTER SUMMARY


Biochemistry_Lippinco

Biochemistry_Lippinco. The pentose phosphate pathway includes an irreversible oxidative phase followed by a series of reversible sugar–phosphate interconversions (Fig. 13.14). No ATP is directly consumed or produced in the pathway. The reduced nicotinamide adenine dinucleotide phosphate (NADPH)-producing oxidative portion of the pathway is important in providing reducing equivalents for reductive biosynthesis and detoxification reactions. In this part of the pathway, glucose 6-phosphate is irreversibly converted to ribulose 5-phosphate, and two NADPH are produced. The regulated step is catalyzed by glucose 6-phosphate dehydrogenase (G6PD), which is strongly inhibited by a rise in the NADPH/NADP+ ratio. Reversible nonoxidative reactions interconvert sugars. This part of the pathway converts ribulose 5-phosphate to ribose 5-phosphate, required for nucleotide and nucleic acid synthesis, or to fructose 6-phosphate and glyceraldehyde 3-phosphate (glycolytic intermediates). NADPH is a source of reducing


Biochemistry_Lippinco

Biochemistry_Lippinco. to ribose 5-phosphate, required for nucleotide and nucleic acid synthesis, or to fructose 6-phosphate and glyceraldehyde 3-phosphate (glycolytic intermediates). NADPH is a source of reducing equivalents in reductive biosynthesis, such as the production of fatty acids in liver, adipose tissue, and the mammary gland; cholesterol in the liver; and steroid hormones in the placenta, ovaries, testes, and adrenal cortex. It is also required by red blood cells (RBC) for hydrogen peroxide reduction. Reduced glutathione (G-SH) is used by glutathione peroxidase to reduce the peroxide to water. The oxidized glutathione (G-S-S-G) produced is reduced by glutathione reductase, using NADPH as the source of electrons. NADPH provides reducing equivalents for the mitochondrial cytochrome P450 monooxygenase system, which is used in steroid hormone synthesis in steroidogenic tissue, bile acid synthesis in the liver, and vitamin D activation in the liver and kidneys. The microsomal system uses NADPH to


Biochemistry_Lippinco

Biochemistry_Lippinco. system, which is used in steroid hormone synthesis in steroidogenic tissue, bile acid synthesis in the liver, and vitamin D activation in the liver and kidneys. The microsomal system uses NADPH to detoxify foreign compounds (xenobiotics), such as drugs and a variety of pollutants. NADPH provides the reducing equivalents for phagocytes in the process of eliminating invading microorganisms. NADPH oxidase uses molecular oxygen (O2) and electrons from NADPH to produce superoxide radicals, which, in turn, can be converted to peroxide by superoxide dismutase. Myeloperoxidase catalyzes the formation of bactericidal hypochlorous acid from peroxide and chloride ions. Rare genetic defects in NADPH oxidase cause chronic granulomatous disease characterized by severe, persistent, infections and granuloma formation. NADPH is required for the synthesis of nitric oxide (NO), an important free radical gas that causes vasodilation by relaxing vascular smooth muscle, acts as a neurotransmitter, prevents


Biochemistry_Lippinco

Biochemistry_Lippinco. formation. NADPH is required for the synthesis of nitric oxide (NO), an important free radical gas that causes vasodilation by relaxing vascular smooth muscle, acts as a neurotransmitter, prevents platelet aggregation, and helps mediate macrophage bactericidal activity. NO is made from arginine and O2 by three different NADPH-dependent NO synthases (NOS). The endothelial (eNOS) and neuronal (nNOS) isozymes constantly produce very low levels of NO for vasodilation and neurotransmission, respectively. The inducible isozyme (iNOS) produces large amounts of NO for defense against pathogens. G6PD deficiency impairs the ability of the cell to form the NADPH that is essential for the maintenance of the G-SH pool. The cells most affected are RBC because they do not have additional sources of NADPH. G6PD deficiency is an X-linked disease characterized by hemolytic anemia caused by the production of free radicals and peroxides following administration of oxidant drugs, ingestion of fava beans,


Biochemistry_Lippinco

Biochemistry_Lippinco. G6PD deficiency is an X-linked disease characterized by hemolytic anemia caused by the production of free radicals and peroxides following administration of oxidant drugs, ingestion of fava beans, or severe infections. The extent of the anemia depends on the amount of residual enzyme. Class I variants, the most severe (and least common), are associated with chronic nonspherocytic hemolytic anemia. Babies with G6PD deficiency may experience neonatal jaundice.


Biochemistry_Lippinco

Biochemistry_Lippinco. Choose the ONE best answer. 3.1. In preparation for a trip to an area of India where chloroquine-resistant malaria is endemic, a young man is given primaquine prophylactically. Soon thereafter, he develops a hemolytic condition due to a deficiency in glucose 6-phosphate dehydrogenase. A less-than-normal level of which of the following is a consequence of the enzyme deficiency and the underlying cause of the hemolysis? A. Glucose 6-phosphate B. Oxidized form of nicotinamide adenine dinucleotide C. Reduced form of glutathione D. Ribose 5-phosphate


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Glucose 6-phosphate B. Oxidized form of nicotinamide adenine dinucleotide C. Reduced form of glutathione D. Ribose 5-phosphate Correct answer = C. Glutathione (G-SH) is essential for red cell integrity and is maintained in this reduced (functional) form by nicotinamide adenine dinucleotide phosphate (NADPH)-dependent glutathione reductase. The NADPH is from the oxidative portion of the pentose phosphate pathway. Individuals with a deficiency of the regulated enzyme of this pathway, glucose 6-phosphate dehydrogenase (G6PD), have a decreased ability to generate NADPH and, therefore, a decreased ability to keep G-SH reduced. When treated with an oxidant drug such as primaquine, some patients with G6PD deficiency develop a hemolytic anemia. Primaquine does not affect glucose 6phosphate levels. Nicotinamide adenine dinucleotide (NAD[H]) is neither produced by the pathway nor used as a coenzyme by G-SH reductase. A decrease in ribose 5-phosphate does not cause hemolysis.


Biochemistry_Lippinco

Biochemistry_Lippinco. 3.2. Septic shock, a state of acute circulatory failure characterized by persistent arterial hypotension (low blood pressure) and inadequate organ perfusion refractory to fluid resuscitation, results from a severe inflammatory response to bacterial infection. It has a high mortality rate and is associated with changes in the level of nitric oxide. Which statement concerning septic shock is most likely correct? A. Activation of endothelial nitric oxide synthase causes an increase in nitric oxide. B. High mortality is the result of the long half-life of nitric oxide. C. Lysine, the nitrogen source for nitric oxide synthesis, is deaminated by bacteria. D. Overproduction of nitric oxide by a calcium-independent enzyme is the cause of the hypotension.


Biochemistry_Lippinco

Biochemistry_Lippinco. C. Lysine, the nitrogen source for nitric oxide synthesis, is deaminated by bacteria. D. Overproduction of nitric oxide by a calcium-independent enzyme is the cause of the hypotension. Correct answer = D. Overproduction of short-lived (not long-lived) nitric oxide (NO) by calcium-independent, inducible nitric oxide synthase (iNOS) results in excessive vasodilation leading to hypotension. The endothelial enzyme (eNOS) is constitutive and produces low levels of NO at a consistent rate. NOS use arginine, not lysine, as the source of the nitrogen.


Biochemistry_Lippinco

Biochemistry_Lippinco. 3.3. An individual who has recently been prescribed a drug (atorvastatin) to lower cholesterol levels is advised to limit consumption of grapefruit juice, because high intake of the juice reportedly results in an increased level of the drug in the blood, increasing the risk of side effects. Atorvastatin is a substrate for the cytochrome P450 enzyme CYP3A4, and grapefruit juice inhibits the enzyme. Which statement concerning CYP enzymes is most likely correct? They: A. accept electrons from reduced nicotinamide adenine dinucleotide. B. catalyze the hydroxylation of hydrophobic molecules. C. differ from nitric oxide synthase in that they contain heme. D. function in association with an oxidase.


Biochemistry_Lippinco

Biochemistry_Lippinco. B. catalyze the hydroxylation of hydrophobic molecules. C. differ from nitric oxide synthase in that they contain heme. D. function in association with an oxidase. Correct answer = B. The CYP enzymes hydroxylate hydrophobic compounds, making them more water soluble. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) from the pentose phosphate pathway is the electron donor. Both the CYP enzymes and the nitric oxide synthase isozymes contain heme. 3.4. In male patients who are hemizygous for X-linked glucose 6-phosphate dehydrogenase deficiency, pathophysiologic consequences are more apparent in red blood cells (RBC) than in other cells such as in the liver. Which one of the following provides the most reasonable explanation for this different response? A. Excess glucose 6-phosphate in the liver, but not in RBC, can be channeled to glycogen, thereby averting cellular damage.


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Excess glucose 6-phosphate in the liver, but not in RBC, can be channeled to glycogen, thereby averting cellular damage. B. Liver cells, in contrast to RBC, have alternative mechanisms for supplying the reduced nicotinamide adenine dinucleotide phosphate required for maintaining cell integrity. C. Because RBC do not have mitochondria, production of ATP required to maintain cell integrity depends exclusively on the shunting of glucose 6-phosphate to the pentose phosphate pathway. D. In RBC, in contrast to liver cells, glucose 6-phosphatase activity decreases the level of glucose 6-phosphate, resulting in cell damage.


Biochemistry_Lippinco

Biochemistry_Lippinco. D. In RBC, in contrast to liver cells, glucose 6-phosphatase activity decreases the level of glucose 6-phosphate, resulting in cell damage. Correct answer = B. Cellular damage is directly related to decreased ability of the cell to regenerate reduced glutathione, for which large amounts of reduced nicotinamide adenine dinucleotide phosphate (NADPH) are needed, and RBC have no means other than the pentose phosphate pathway of generating NADPH. It is decreased product (NADPH), not increased substrate (glucose 6phosphate), that is the problem. RBC do not have glucose 6-phosphatase. The pentose phosphate pathway does not generate ATP. 3.5. An essential coenzyme for several enzymes of metabolism is derived from the vitamin thiamine. Measurement of the activity of what enzyme in red blood cells could be used to determine thiamine status in the body?


Biochemistry_Lippinco

Biochemistry_Lippinco. Red blood cells do not have mitochondria and, so, do not contain mitochondrial enzymes such as pyruvate dehydrogenase that require the thiamine-derived coenzyme thiamine pyrophosphate (TPP). However, they do contain the cytosolic TPP-requiring transketolase, whose activity is used clinically to assess thiamine status. Glycosaminoglycans, Proteoglycans, and Glycoproteins 14 For additional ancillary materials related to this chapter, please visit thePoint. I. GLYCOSAMINOGLYCAN OVERVIEW


Biochemistry_Lippinco

Biochemistry_Lippinco. I. GLYCOSAMINOGLYCAN OVERVIEW Glycosaminoglycans (GAG) are large complexes of negatively charged heteropolysaccharide chains. They are generally associated with a small amount of protein (core protein), forming proteoglycans, which typically consist of up to 95% carbohydrate. GAG have the special ability to bind large amounts of water, thereby producing the gel-like matrix that forms the basis of the body’s ground substance, which, along with fibrous structural proteins such as collagen, elastin, and fibrillin-1, and adhesive proteins such as fibronectin, makes up the extracellular matrix (ECM). The hydrated GAG serve as a flexible support for the ECM, interacting with the structural and adhesive proteins, and as a molecular sieve, influencing movement of materials through the ECM. The viscous, lubricating properties of mucous secretions also result from the presence of GAG, which led to the original naming of these compounds as mucopolysaccharides. II. STRUCTURE


Biochemistry_Lippinco

Biochemistry_Lippinco. II. STRUCTURE GAG are long, unbranched, heteropolysaccharide chains composed of a repeating disaccharide unit [acidic sugar–amino sugar]n (Fig. 14.1). [Note: A single exception is keratan sulfate, which contains galactose rather than an acidic sugar.] The amino sugar is either D-glucosamine or D-galactosamine, in which the amino group is usually acetylated, thus eliminating its positive charge. The amino sugar may also be sulfated on carbon 4 or 6 or on a nonacetylated nitrogen. The acidic sugar is either D-glucuronic acid or its C-5 epimer Liduronic acid (Fig. 14.2). These uronic sugars contain carboxyl groups that are negatively charged at physiologic pH and, together with the sulfate groups (−SO42−), give GAG their strongly negative nature. A. Structure–function relationship


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Structure–function relationship Because of the high concentration of negative charges, these heteropolysaccharide chains tend to be extended in solution. They repel each other and are surrounded by a shell of water molecules. When brought together, they slide past each other, much as two magnets with the same polarity seem to slide past each other. This produces the slippery consistency of mucous secretions and synovial fluid. When a solution of GAG is compressed, the water is squeezed out, and the GAG are forced to occupy a smaller volume. When the compression is released, the GAG spring back to their original, hydrated volume because of the repulsion of their negative charges. This property contributes to the resilience of synovial fluid and the vitreous humor of the eye (Fig. 14.3). B. Classification


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Classification The six major types of GAG are divided according to monomeric composition, type of glycosidic linkages, and degree and location of sulfate units. The structure of the GAG and their distribution in the body is illustrated in Figure 14.4. All GAG, except for hyaluronic acid, are sulfated and are found covalently attached to protein, forming proteoglycan monomers. C. Proteoglycans Proteoglycans are found in the ECM and on the outer surface of cells. 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. C. Proteoglycans Proteoglycans are found in the ECM and on the outer surface of cells. 1. Monomer structure: A proteoglycan monomer found in cartilage consists of a core protein to which up to 100 linear chains of GAG are covalently attached. These chains, which may each be composed of up to 200 disaccharide units, extend out from the core protein and remain separated from each other because of charge repulsion. The resulting structure resembles a bottle brush (Fig. 14.5). In cartilage proteoglycans, the species of GAG include chondroitin sulfate and keratan sulfate. [Note: Proteoglycans are grouped into gene families that encode core proteins with common structural features. The aggrecan family (aggrecan, versican, neurocan, and brevican), abundant in cartilage, is an example.] 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. GAG–protein linkage: This covalent linkage is most commonly through a trihexoside (galactose-galactose-xylose) and a serine residue in the protein. An O-glycosidic bond (see p. 86) is formed between the xylose and the hydroxyl group of the serine (Fig. 14.6). 3. Aggregate formation: Many proteoglycan monomers can associate with one molecule of hyaluronic acid to form proteoglycan aggregates. The association is not covalent and occurs primarily through ionic interactions between the core protein and the hyaluronic acid. The association is stabilized by additional small proteins called link proteins (Fig. 14.7). III. SYNTHESIS


Biochemistry_Lippinco

Biochemistry_Lippinco. III. SYNTHESIS The heteropolysaccharide chains are elongated by the sequential addition of alternating acidic and amino sugars donated primarily by their uridine diphosphate (UDP) derivatives. The reactions are catalyzed by a family of specific glycosyltransferases. Because GAG are produced for export from the cell, their synthesis occurs primarily in the Golgi and not in the cytosol. A. Amino sugar synthesis Amino sugars are essential components of glycoconjugates such as proteoglycans, glycoproteins, and glycolipids. The synthetic pathway of amino sugars (hexosamines) is very active in connective tissues, where as much as 20% of glucose flows through this pathway.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. N-Acetylglucosamine and N-acetylgalactosamine: The monosaccharide fructose 6-phosphate is the precursor of N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc). A hydroxyl group on the fructose is replaced by the amide nitrogen of a glutamine, and the glucosamine 6phosphate product gets acetylated, isomerized, and activated, producing the nucleotide sugar UDP-GlcNAc (Fig. 14.8). UDP-GalNAc is generated by the epimerization of UDP-GlcNAc. It is these nucleotide sugar forms of the amino sugars that are used to elongate the carbohydrate chains. pyrophosphate.


Biochemistry_Lippinco

Biochemistry_Lippinco. pyrophosphate. 2. N-Acetylneuraminic acid: NANA, a nine-carbon, acidic monosaccharide (see Fig. 17.15, p. 209), is a member of the family of sialic acids, each of which is acylated at a different site. These compounds are usually found as terminal carbohydrate residues of oligosaccharide side chains of glycoproteins, of glycolipids, or, less frequently, of GAG. N-Acetylmannosamine 6-phosphate (derived from fructose 6-phosphate) and phosphoenolpyruvate (an intermediate in glycolysis; see p. 102) are the immediate sources of the carbons and nitrogens for NANA synthesis (see Fig. 14.8). Before NANA can be added to a growing oligosaccharide, it must be activated to cytidine monophosphate (CMP)NANA by reacting with cytidine triphosphate (CTP). CMP-NANA synthetase catalyzes the reaction. [Note: CMP-NANA is the only nucleotide sugar in human metabolism in which the carrier nucleotide is a monophosphate rather than a diphosphate.] B. Acidic sugar synthesis


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Acidic sugar synthesis D-Glucuronic acid, whose structure is that of glucose with an oxidized carbon 6 (−CH2OH → −COOH), and its C-5 epimer, L-iduronic acid, are essential components of GAG. Glucuronic acid is also required for the detoxification of lipophilic compounds, such as bilirubin (see p. 282), steroids (see p. 240), and many drugs, including the statins (see p. 224), because conjugation with glucuronate (glucuronidation) increases water solubility. In plants and mammals (other than guinea pigs and primates, including humans), glucuronic acid is a precursor of ascorbic acid (vitamin C) as shown in Figure 14.9. This uronic acid pathway also provides a mechanism by which dietary D-xylulose can enter the central metabolic pathways.


Biochemistry_Lippinco

Biochemistry_Lippinco. C) as shown in Figure 14.9. This uronic acid pathway also provides a mechanism by which dietary D-xylulose can enter the central metabolic pathways. 1. Glucuronic acid: Glucuronic acid can be obtained in small amounts from the diet and from the lysosomal degradation of GAG. It also can be synthesized by the uronic acid pathway, in which glucose 1-phosphate reacts with uridine triphosphate (UTP) and is converted to UDP-glucose. Oxidation of UDP-glucose produces UDP-glucuronic acid, the form that supplies glucuronic acid for GAG synthesis and glucuronidation (Fig. 14.10). The end product of glucuronic acid metabolism in humans is Dxylulose 5-phosphate, which can enter the pentose phosphate pathway and produce the glycolytic intermediates glyceraldehyde 3-phosphate and fructose 6-phosphate (see Fig. 14.9; see also Fig. 13.2, p. 146).


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. l-Iduronic acid: Synthesis of L-iduronic acid occurs after D-glucuronic acid has been incorporated into the carbohydrate chain. Uronosyl 5epimerase causes epimerization of the D-to the L-sugar. C. Core protein synthesis The core protein is made by ribosomes on the rough endoplasmic reticulum (RER), enters the RER lumen, and then moves to the Golgi, where it is glycosylated by membrane-bound glycosyltransferases. D. Carbohydrate chain synthesis Carbohydrate chain formation is initiated by synthesis of a short linker on the core protein on which carbohydrate chain synthesis will occur. The most common linker is a trihexoside formed by the transfer of a xylose from UDP-xylose to the hydroxyl group of a serine (or threonine) catalyzed by xylosyltransferase. Two galactose molecules are then added, completing the trihexoside. This is followed by sequential addition of alternating acidic and amino sugars (Fig. 14.11) and epimerization of some D-glucuronyl to Liduronyl residues.


Biochemistry_Lippinco

Biochemistry_Lippinco. E. Sulfate group addition Sulfation of a GAG occurs after the monosaccharide to be sulfated has been incorporated into the growing carbohydrate chain. The source of the sulfate is 3´-phosphoadenosyl-5´-phosphosulfate ([PAPS] a molecule of adenosine monophosphate with a sulfate group attached to the 5´-phosphate; see Fig. 17.16, p. 210). The sulfation reaction is catalyzed by sulfotransferases. Synthesis of the sulfated GAG chondroitin sulfate is shown in Figure 14.11. [Note: PAPS is also the sulfur donor in glycosphingolipid synthesis (see p. 210).] A defect in the sulfation of the growing GAG chains results in one of several autosomal-recessive disorders, the chondrodystrophies, which affect the proper development and maintenance of the skeletal system. IV. DEGRADATION


Biochemistry_Lippinco

Biochemistry_Lippinco. IV. DEGRADATION GAG are degraded in lysosomes, which contain hydrolytic enzymes that are most active at a pH of ~5. Therefore, as a group, these enzymes are called acid hydrolases. [Note: The low pH optimum is a protective mechanism that prevents the enzymes from destroying the cell should leakage occur into the cytosol where the pH is neutral.] The half-lives of GAG vary from minutes to months and are influenced by the type of GAG and its location in the body. A. GAG phagocytosis Because GAG are extracellular or cell-surface compounds, they must first be engulfed by invagination of the cell membrane (phagocytosis), forming a vesicle inside of which are the GAG to be degraded. This vesicle then fuses with a lysosome, forming a single digestive vesicle in which the GAG are efficiently degraded (see p. 150 for a discussion of phagocytosis). B. Lysosomal degradation


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Lysosomal degradation The lysosomal degradation of GAG requires a large number of acid hydrolases for complete digestion. First, the polysaccharide chains are cleaved by endoglycosidases, producing oligosaccharides. Further degradation of the oligosaccharides occurs sequentially from the nonreducing end (see p. 127) of each chain, the last group (sulfate or sugar) added during synthesis being the first group removed (by sulfatases or exoglycosidases). Examples of some of these enzymes and the bonds they hydrolyze are shown in Figure 14.12. [Note: Endo-and exoglycosidases are also involved in the lysosomal degradation of glycoproteins (see p. 170) and glycolipids (see p. 210). Deficiencies in these enzymes result in the accumulation of partially degraded carbohydrates, causing tissue damage.] glucosamine; S = sulfate.


Biochemistry_Lippinco

Biochemistry_Lippinco. Multiple sulfatase deficiency (Austin disease) is a rare lysosomal storage disease in which all sulfatases are nonfunctional because of a defect in the formation of formylglycine, an amino acid derivative required at the active site for enzymic activity to occur. V. MUCOPOLYSACCHARIDOSES


Biochemistry_Lippinco

Biochemistry_Lippinco. The mucopolysaccharidoses are hereditary diseases (approximately 1:25,000 live births) caused by a deficiency of any one of the lysosomal hydrolases normally involved in the degradation of heparan sulfate and/or dermatan sulfate (see Fig. 14.12). They are progressive disorders characterized by lysosomal accumulation of GAG in various tissues, causing a range of symptoms, such as skeletal and ECM deformities, and intellectual disability. All are autosomal-recessive disorders except Hunter syndrome, which is X linked. Children who are homozygous for any one of these diseases are apparently normal at birth and then gradually deteriorate. In severe deficiencies, death occurs in childhood. There currently is no cure. Incomplete lysosomal degradation of GAG results in the presence of oligosaccharides in the urine. These fragments can be used to diagnose the specific mucopolysaccharidosis by identifying the structure present on the nonreducing end of the oligosaccharide, because that residue


Biochemistry_Lippinco

Biochemistry_Lippinco. in the urine. These fragments can be used to diagnose the specific mucopolysaccharidosis by identifying the structure present on the nonreducing end of the oligosaccharide, because that residue would have been the substrate for the missing enzyme. Diagnosis is confirmed by measuring the patient’s cellular level of the lysosomal hydrolases. Bone marrow and cord blood transplants, in which transplanted macrophages produce the enzymes that degrade GAG, have been used to treat Hurler and Hunter syndromes, with limited success. Enzyme replacement therapy is available for both syndromes but does not prevent neurologic damage.


Biochemistry_Lippinco

VI. GLYCOPROTEIN OVERVIEW

Biochemistry_Lippinco. VI. GLYCOPROTEIN OVERVIEW


Biochemistry_Lippinco

Biochemistry_Lippinco. Glycoproteins are proteins to which oligosaccharides (glycans) are covalently attached. [Note: Glycosylation is the most common posttranslational modification of proteins.] They differ from the proteoglycans in several important ways. Glycoproteins contain highly variable amounts of carbohydrate but typically less than that of proteoglycans. For example, the glycoprotein immunoglobulin G (IgG) contains <4% of its mass as carbohydrate, whereas the proteoglycan aggrecan contains >80%. In glycoproteins, the glycan is relatively short (usually two to ten sugar residues in length, although it can be longer); does not contain repeating disaccharide units and, consequently, is structurally diverse; is often branched instead of linear; and may or may not be negatively charged. Membrane-bound glycoproteins participate in a broad range of cellular phenomena, including cell-surface recognition (by other cells, hormones, and viruses), cell-surface antigenicity (such as the blood group antigens),


Biochemistry_Lippinco

Biochemistry_Lippinco. participate in a broad range of cellular phenomena, including cell-surface recognition (by other cells, hormones, and viruses), cell-surface antigenicity (such as the blood group antigens), and as components of the ECM and of the mucins of the gastrointestinal and urogenital tracts, where they act as protective biologic lubricants. In addition, almost all of the globular proteins present in human plasma are glycoproteins, although albumin is an exception. Figure 14.13 summarizes some glycoprotein functions.


Biochemistry_Lippinco

Biochemistry_Lippinco. VII. OLIGOSACCHARIDE STRUCTURE The oligosaccharide (glycan) components of glycoproteins are generally branched heteropolymers composed primarily of D-hexoses, with the addition in some cases of neuraminic acid (a nonose) and of L-fucose, a 6-deoxyhexose. A. Carbohydrate–protein linkage The glycan may be attached to the protein through an N-or an O-glycosidic link (see p. 86). In the former case, the sugar chain is attached to the amide group of an asparagine side chain and, in the latter case, to the hydroxyl group of either a serine or threonine side chain. [Note: In the case of collagen, there is an O-glycosidic linkage between galactose or glucose and the hydroxyl group of hydroxylysine (see p. 47).] B. N-and O-Linked oligosaccharides A glycoprotein may contain only one type of glycosidic linkage (N or O linked) or may have both types within the same molecule. 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. B. N-and O-Linked oligosaccharides A glycoprotein may contain only one type of glycosidic linkage (N or O linked) or may have both types within the same molecule. 1. O-Linked: The O-linked glycans may have one or more of a wide variety of sugars arranged in either a linear or a branched pattern. Many are found in extracellular glycoproteins or as membrane glycoprotein components. For example, O-linked oligosaccharides on the surface of red blood cells help provide the ABO blood group determinants. If the terminal sugar on the glycan is GalNAc, the blood group is A. If it is galactose, the blood group is B. If neither GalNAc nor galactose is present, the blood group is O. 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. N-Linked: The N-linked glycans fall into two broad classes: complex oligosaccharides and high-mannose oligosaccharides. Both contain the same pentasaccharide core shown in Figure 14.14, but the complex oligosaccharides contain a diverse group of additional sugars, for example, GlcNAc, GalNAc, L-fucose, and NANA, whereas the high mannose oligosaccharides contain primarily mannose. VIII. GLYCOPROTEIN SYNTHESIS Proteins destined to function in the cytoplasm are synthesized on free cytosolic ribosomes. However, proteins, including glycoproteins, that are destined for cellular membranes, lysosomes, or to be exported from the cell, are synthesized on ribosomes attached to the RER. These proteins contain specific signal sequences that act as molecular addresses, targeting the proteins to their proper destinations. An N-terminal hydrophobic sequence initially directs these proteins to the RER, allowing the growing polypeptide to be extruded into the lumen (see p.


Biochemistry_Lippinco

Biochemistry_Lippinco. 459). The proteins are then transported via secretory vesicles to the Golgi, which acts as a sorting center (Fig. 14.15). In the Golgi, those glycoproteins that are to be secreted from the cell (or are targeted for lysosomes) are packaged into vesicles that fuse with the cell (or lysosomal) membrane and release their contents. Those that are destined to become components of the cell membrane are integrated into the Golgi membrane, which buds off, forming vesicles that add their membrane-bound glycoproteins to the cell membrane. [Note: Therefore, the membrane glycoproteins are oriented with the carbohydrate portion on the outside of the cell (see Fig. 14.15).] A.


Biochemistry_Lippinco

Biochemistry_Lippinco. A. The precursors of the carbohydrate components of glycoproteins are nucleotide sugars, which include UDP-glucose, UDP-galactose, UDP-GlcNAc, and UDP-GalNAc. In addition, guanosine diphosphate (GDP)mannose, GDP-L-fucose (which is synthesized from GDP-mannose), and CMP-NANA may donate sugars to the growing chain. [Note: When the acidic NANA is present, the oligosaccharide has a negative charge at physiologic pH.] The oligosaccharides are covalently attached to the side chains of specific amino acids in the protein, where the three-dimensional structure of the protein determines whether or not a specific amino acid is glycosylated. B. O-Linked glycoprotein synthesis


Biochemistry_Lippinco

Biochemistry_Lippinco. B. O-Linked glycoprotein synthesis Synthesis of the O-linked glycoproteins is very similar to that of the GAG (see p. 158). First, the protein to which sugars are to be attached is synthesized on the RER and extruded into its lumen. Glycosylation begins with the transfer of GalNAc (from UDP-GalNAc) to the hydroxyl group of a specific serine or threonine residues. The glycosyltransferases responsible for the stepwise synthesis (from individual sugars) of the oligosaccharides are bound to the membranes of the Golgi. They act in a specific order, without using a template as is required for DNA, ribonucleic acid (RNA), and protein synthesis (see Unit VII), but instead by recognizing the actual structure of the growing oligosaccharide as the appropriate substrate. C. N-Linked glycoprotein synthesis


Biochemistry_Lippinco

Biochemistry_Lippinco. C. N-Linked glycoprotein synthesis Synthesis of N-linked glycoproteins occurs in the lumen of the RER and requires the participation of the phosphorylated form of dolichol (dolichol pyrophosphate), a lipid of the RER membrane (Fig. 14.16). The initial product is processed in the RER and Golgi. = terminal group (fucose or Nacetylneuraminic acid); mRNA = messenger RNA; Asn = asparagine.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. Dolichol-linked oligosaccharide synthesis: As with the O-linked glycoproteins, the protein is synthesized on the RER and enters its lumen. However, it does not become glycosylated with individual sugars. Instead, a lipid-linked oligosaccharide is first constructed. This consists of dolichol (an RER membrane lipid made from an intermediate of cholesterol synthesis; see p. 221) attached through a pyrophosphate linkage to an oligosaccharide containing GlcNAc, mannose, and glucose. The sugars to be added sequentially to the dolichol by membrane-bound glycosyltransferases are first GlcNAc, followed by mannose and glucose (see Fig. 14.16). The entire 14-sugar oligosaccharide is then transferred from dolichol to the amide nitrogen of an asparagine residue in the protein to be glycosylated by a protein–oligosaccharide transferase present in the RER. [Note: Tunicamycin inhibits N-linked glycosylation.]


Biochemistry_Lippinco

Biochemistry_Lippinco. Congenital disorders of glycosylation (CDG) are syndromes caused primarily by defects in the N-linked glycosylation of proteins, either oligosaccharide assembly (type I) or processing (type II). 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. N-Linked oligosaccharide processing: After addition to the protein, the N-linked oligosaccharide is processed by the removal of specific mannosyl and glucosyl residues as the glycoprotein moves through the RER. Finally, the oligosaccharide chains are completed in the Golgi by addition of a variety of sugars (for example, GlcNAc, GalNAc, and additional mannoses and then fucose or NANA as terminal groups) to produce a complex glycoprotein. Alternatively, they are not processed further, leaving branched, mannose-containing chains in a high-mannose glycoprotein (see Fig. 14.16). The ultimate fate of N-linked glycoproteins is the same as that of the O-linked glycoproteins (for example, they can be released by the cell or become part of a cell membrane). In addition, N-linked glycoproteins can be targeted to the lysosomes. [Note: Nonenzymatic glycosylation of proteins is known as glycation (see p. 33).] 3.


Biochemistry_Lippinco

Biochemistry_Lippinco. Lysosomal enzymes: N-Linked glycoproteins being processed in the Golgi can be phosphorylated on carbon 6 of one or more mannosyl residues. UDP-GlcNAc provides the phosphate in a reaction catalyzed by a phosphotransferase. Receptors, located in the Golgi membrane, bind the mannose 6-phosphate residues of these proteins, which are then packaged into vesicles and sent to the lysosomes (Fig. 14.17). I-Cell disease is a rare lysosomal storage disease in which the phosphotransferase is deficient. This causes the proteins to be secreted, rather than being targeted to lysosomes. Consequently, the acid hydrolases normally found in the lysosomal matrix are absent, resulting in an accumulation of the substrates for these missing enzymes. [Note: I-Cell disease is so named because of the large inclusion bodies seen in cells of patients with this disease.] In addition, high amounts of lysosomal enzymes are found in the patient’s plasma and urine, indicating that the targeting process to lysosomes


Biochemistry_Lippinco

Biochemistry_Lippinco. bodies seen in cells of patients with this disease.] In addition, high amounts of lysosomal enzymes are found in the patient’s plasma and urine, indicating that the targeting process to lysosomes (rather than the synthetic pathway of these enzymes) is deficient. I-Cell disease is characterized by skeletal abnormalities, restricted joint movement, coarse (dysmorphic) facial features, and severe psychomotor impairment. [Note:


Biochemistry_Lippinco

Biochemistry_Lippinco. Because I-cell disease has features in common with the mucopolysaccharidoses and sphingolipidoses (see p. 210), it is termed a mucolipidosis (type II).] Currently, there is no cure, and death from cardiopulmonary complications usually occurs in early childhood. IX. LYSOSOMAL GLYCOPROTEIN DEGRADATION


Biochemistry_Lippinco

Biochemistry_Lippinco. IX. LYSOSOMAL GLYCOPROTEIN DEGRADATION Degradation of glycoproteins is similar to that of the GAG (see p. 163). The lysosomal acid hydrolases are each generally specific for the removal of one component of the glycoprotein. They are primarily exoenzymes that remove their respective groups in the reverse order of their incorporation (last on, first off). If any one degradative enzyme is missing, degradation by the other exoenzymes cannot continue. A group of very rare autosomal-recessive diseases called the glycoprotein storage diseases (oligosaccharidoses), caused by a deficiency of any one of the degradative enzymes, results in accumulation of partially degraded structures in the lysosomes. For example, α-mannosidosis type 3 is a severe, progressive, fatal deficiency of the enzyme α-mannosidase. Presentation is similar to Hurler syndrome, but immune deficiency is also seen. Mannose-rich oligosaccharide fragments appear in the urine. Diagnosis is by enzyme assay. X. CHAPTER SUMMARY


Biochemistry_Lippinco

Biochemistry_Lippinco. Glycosaminoglycans (GAG) are long, negatively charged, unbranched, heteropolysaccharide chains generally composed of a repeating disaccharide unit [acidic sugar–amino sugar]n (Fig. 14.18). The amino sugar is either D-glucosamine or D-galactosamine in which the amino group is usually acetylated, thus eliminating its positive charge. The amino sugar may also be sulfated on carbon 4 or 6 or on a nonacetylated nitrogen. The acidic sugar is either D-glucuronic acid or its C-5 epimer L-iduronic acid. GAG bind large amounts of water, thereby producing the gel-like matrix that forms the basis of the body’s ground substance. The viscous, lubricating properties of mucous secretions are also caused by the presence of GAG, which led to the original naming of these compounds as mucopolysaccharides. There are six major types of GAG, including chondroitin 4-and 6-sulfates, keratan sulfate, dermatan sulfate, heparin, heparan sulfate, and hyaluronic acid. All GAG, except hyaluronic acid, are found


Biochemistry_Lippinco

Biochemistry_Lippinco. There are six major types of GAG, including chondroitin 4-and 6-sulfates, keratan sulfate, dermatan sulfate, heparin, heparan sulfate, and hyaluronic acid. All GAG, except hyaluronic acid, are found covalently attached to a core protein, forming proteoglycan monomers. Many proteoglycan monomers associate with a molecule of hyaluronic acid to form proteoglycan aggregates. GAG are synthesized in the Golgi. The polysaccharide chains are elongated by the sequential addition of alternating acidic and amino sugars, donated by their UDP derivatives. D-Glucuronate may be epimerized to L-iduronate. The last step in synthesis is sulfation of some of the amino sugars. The source of the sulfate is 3′-phosphoadenosyl-5′-phosphosulfate (PAPS). The completed proteoglycans are secreted into the extracellular matrix (ECM) or remain associated with the outer surface of cells. GAG are degraded by lysosomal acid hydrolases. They are first broken down to oligosaccharides, which are degraded sequentially


Biochemistry_Lippinco

Biochemistry_Lippinco. matrix (ECM) or remain associated with the outer surface of cells. GAG are degraded by lysosomal acid hydrolases. They are first broken down to oligosaccharides, which are degraded sequentially from the nonreducing end of each chain. A deficiency of any one of the hydrolases results in a mucopolysaccharidosis. These are hereditary disorders in which GAG accumulate in tissues, causing symptoms such as skeletal and ECM deformities and intellectual disability. Examples of these genetic diseases include Hunter (X-linked) and Hurler syndromes. Glycoproteins are proteins to which oligosaccharides (glycans) are covalently attached. They differ from the proteoglycans in that the length of the glycoprotein’s carbohydrate chain is relatively short (usually two to ten sugar residues long, although it can be longer), may be branched, and does not contain serial disaccharide units. Membrane-bound glycoproteins participate in a broad range of cellular phenomena, including cell-surface recognition


Biochemistry_Lippinco

Biochemistry_Lippinco. it can be longer), may be branched, and does not contain serial disaccharide units. Membrane-bound glycoproteins participate in a broad range of cellular phenomena, including cell-surface recognition (by other cells, hormones, and viruses), cell-surface antigenicity (such as the blood group antigens), and as components of the ECM and of the mucins of the gastrointestinal and urogenital tracts, where they act as protective biologic lubricants. In addition, almost all of the globular proteins present in human plasma are glycoproteins. Glycoproteins are synthesized in the rough endoplasmic reticulum (RER) and the Golgi. The precursors of the carbohydrate components of glycoproteins are nucleotide sugars. O-Linked glycoproteins are synthesized in the Golgi by the sequential transfer of sugars from their nucleotide carriers to the hydroxyl group of a serine or threonine residue in the protein. N-Linked glycoproteins are synthesized by the transfer of a preformed oligosaccharide from its


Biochemistry_Lippinco

Biochemistry_Lippinco. from their nucleotide carriers to the hydroxyl group of a serine or threonine residue in the protein. N-Linked glycoproteins are synthesized by the transfer of a preformed oligosaccharide from its RER membrane lipid carrier, dolichol pyrophosphate, to the amide N of an asparagine residue in the protein. They contain varying amounts of mannose. A deficiency in the phosphotransferase that phosphorylates mannose residues at carbon 6 in N-linked glycoprotein enzymes destined for the lysosomes results in I-cell disease. Glycoproteins are degraded in lysosomes by acid hydrolases. A deficiency of any one of these enzymes results in a lysosomal glycoprotein storage disease (oligosaccharidosis), resulting in accumulation of partially degraded structures in the lysosome.


Biochemistry_Lippinco

Biochemistry_Lippinco. Choose the ONE best answer. 4.1. Mucopolysaccharidoses are hereditary lysosomal storage diseases. They are caused by: A. defects in the degradation of glycosaminoglycans. B. defects in the targeting of enzymes to lysosomes. C. an increased rate of synthesis of the carbohydrate component of proteoglycans. D. an insufficient rate of synthesis of proteolytic enzymes. E. the synthesis of abnormally small amounts of core proteins. F. the synthesis of heteropolysaccharides with an altered structure. Correct answer = A. The mucopolysaccharidoses are caused by deficiencies in any one of the lysosomal acid hydrolases responsible for the degradation of glycosaminoglycans (not proteins). The enzyme is correctly targeted to the lysosome, so blood levels of the enzyme do not increase, but it is nonfunctional. In these diseases, synthesis of the protein and carbohydrate components of proteoglycans is unaffected, in terms of both structure and amount.


Biochemistry_Lippinco

Biochemistry_Lippinco. 4.2. The presence of the following compound in the urine of a patient suggests a deficiency in which one of the enzymes listed below? A. Galactosidase B. Glucuronidase C. Iduronidase D. Mannosidase E. Sulfatase Correct answer = E. Degradation of glycoproteins follows the rule: last on, first off. Because sulfation is the last step in the synthesis of this sequence, a sulfatase is required for the next step in the degradation of the compound shown. 4.3. An 8-month-old boy with coarse facial features, skeletal abnormalities, and delays in both growth and development is diagnosed with I-cell disease based on his presentation and on histologic and biochemical testing. I-Cell disease is characterized by: A. decreased production of cell surface O-linked glycoproteins. B. elevated levels of acid hydrolases in the blood. C. inability to N-glycosylate proteins. D. increased synthesis of proteoglycans. E. oligosaccharides in the urine.


Biochemistry_Lippinco

Biochemistry_Lippinco. B. elevated levels of acid hydrolases in the blood. C. inability to N-glycosylate proteins. D. increased synthesis of proteoglycans. E. oligosaccharides in the urine. Correct answer = B. I-Cell disease is a lysosomal storage disease caused by deficiency of the phosphotransferase needed for synthesis of the mannose 6phosphate signal that targets acid hydrolases to the lysosomal matrix. This results in secretion of these enzymes from the cell and accumulation of materials within the lysosome because of impaired degradation. None of the other choices relates to I-cell disease or lysosomal function. Oligosaccharides in the urine are characteristic of the muco-and polysaccharidoses but not I-cell disease (a type II mucolipidosis). 4.4. An infant with corneal clouding has dermatan sulfate and heparan sulfate in his urine. Decreased activity of which of the enzymes listed below would confirm the suspected diagnosis of Hurler syndrome? A. α-L-Iduronidase B. α-Glucuronidase


Biochemistry_Lippinco

Biochemistry_Lippinco. A. α-L-Iduronidase B. α-Glucuronidase C. Glycosyltransferase D. Iduronate sulfatase Correct answer = A. Hurler syndrome, a defect in the lysosomal degradation of glycosaminoglycans (GAG) with corneal clouding, is due to a deficiency in αL-iduronidase. β-Glucuronidase is deficient in Sly syndrome, and iduronate sulfatase is deficient in Hunter syndrome. Glycosyltransferases are enzymes of GAG synthesis. 4.5. Distinguish between glycoproteins and proteoglycans. Glycoproteins are proteins to which short, branched, structurally diverse oligosaccharide chains (glycans) are attached. Proteoglycans consist of a core protein to which long, unbranched, glycosaminoglycan (GAG) chains are attached. GAG are large complexes of negatively charged heteropolysaccharides composed of repeating [acidic sugar-amino sugar]n disaccharide units. For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW


Biochemistry_Lippinco

Biochemistry_Lippinco. I. OVERVIEW Lipids are a heterogeneous group of water-insoluble (hydrophobic) organic molecules (Fig. 15.1). Because of their insolubility in aqueous solutions, body lipids are generally found compartmentalized, as in the case of membrane-associated lipids or droplets of triacylglycerol in adipocytes, or transported in blood in association with protein, as in lipoprotein particles (see p. 227) or on albumin. Lipids are a major source of energy for the body, and they also provide the hydrophobic barrier that permits partitioning of the aqueous contents of cells and subcellular structures. Lipids serve additional functions in the body (for example, some fat-soluble vitamins have regulatory or coenzyme functions, and the prostaglandins and steroid hormones play major roles in the control of the body’s homeostasis). Deficiencies or imbalances of lipid metabolism can lead to some of the major clinical problems encountered by physicians, such as atherosclerosis, diabetes, and obesity.


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Biochemistry_Lippinco. II. DIGESTION, ABSORPTION, SECRETION, AND UTILIZATION The average daily intake of lipids by U.S. adults is ~78 g, of which >90% is triacylglycerol ([TAG], formerly called triglyceride [TG]), that consists of three fatty acids (FA) esterified to a glycerol backbone (see Fig. 15.1). The remainder of the dietary lipids consists primarily of cholesterol, cholesteryl esters, phospholipids, and nonesterified (free) FA (FFA). The digestion of dietary lipids begins in the stomach and is completed in the small intestine. The process is summarized in Figure 15.2. A. Digestion in the stomach


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Digestion in the stomach Lipid digestion in the stomach is limited. It is catalyzed by lingual lipase that originates from glands at the back of the tongue and gastric lipase that is secreted by the gastric mucosa. Both enzymes are relatively acid stable, with optimal pH values of 4 to 6. These acid lipases hydrolyze FA from TAG molecules, particularly those containing short-or medium-chainlength (≤12 carbons) FA such as are found in milk fat. Consequently, these lipases play a particularly important role in lipid digestion in infants for whom milk fat is the primary source of calories. They also become important digestive enzymes in individuals with pancreatic insufficiency such as those with cystic fibrosis (CF). Lingual and gastric lipases aid these patients in degrading TAG molecules (especially those with short-to medium-chain FA) despite a near or complete absence of pancreatic lipase (see Section D.1. below). B. Cystic fibrosis


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Cystic fibrosis CF is the most common lethal genetic disease in Caucasians of Northern European ancestry and has a prevalence of ~1:3,300 births in the United States. CF is an autosomal-recessive disorder caused by mutations to the gene for the CF transmembrane conductance regulator (CFTR) protein that functions as a chloride channel on epithelium in the pancreas, lungs, testes, and sweat glands. Defective CFTR results in decreased secretion of chloride and increased uptake of sodium and water. In the pancreas, the depletion of water on the cell surface results in thickened mucus that clogs the pancreatic ducts, preventing pancreatic enzymes from reaching the intestine, thereby leading to pancreatic insufficiency. Treatment includes replacement of these enzymes and supplementation with fat-soluble vitamins. [Note: CF also causes chronic lung infections with progressive pulmonary disease and male infertility.] C. Emulsification in the small intestine


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Biochemistry_Lippinco. C. Emulsification in the small intestine The critical process of dietary lipid emulsification occurs in the duodenum.


Biochemistry_Lippinco

Biochemistry_Lippinco. Emulsification increases the surface area of the hydrophobic lipid droplets so that the digestive enzymes, which work at the interface of the droplet and the surrounding aqueous solution, can act effectively. Emulsification is accomplished by two complementary mechanisms, namely, use of the detergent properties of the conjugated bile salts and mechanical mixing due to peristalsis. Bile salts, made in the liver and stored in the gallbladder, are amphipathic derivatives of cholesterol (see p. 224). Conjugated bile salts consist of a hydroxylated sterol ring structure with a side chain to which a molecule of glycine or taurine is covalently attached by an amide linkage (Fig. 15.3). These emulsifying agents interact with the dietary lipid droplets and the aqueous duodenal contents, thereby stabilizing the droplets as they become smaller from peristalsis and preventing them from coalescing. [Note: See p. 225 for a more complete discussion of bile salt metabolism.]


Biochemistry_Lippinco

Biochemistry_Lippinco. D. Degradation by pancreatic enzymes The dietary TAG, cholesteryl esters, and phospholipids are enzymatically degraded (digested) in the small intestine by pancreatic enzymes, whose secretion is hormonally controlled. 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. Triacylglycerol degradation: TAG molecules are too large to be taken up efficiently by the mucosal cells (enterocytes) of the intestinal villi. Therefore, they are hydrolyzed by an esterase, pancreatic lipase, which preferentially removes the FA at carbons 1 and 3. The primary products of hydrolysis are, thus, a mixture of 2-monoacylglycerol (2-MAG) and FFA (see Fig. 15.2). [Note: Pancreatic lipase is found in high concentrations in pancreatic secretions (2%–3% of the total protein present), and it is highly efficient catalytically, thus insuring that only severe pancreatic deficiency, such as that seen in CF, results in significant malabsorption of fat.] A second protein, colipase, also secreted by the pancreas, binds the lipase at a ratio of 1:1 and anchors it at the lipid–aqueous interface. Colipase restores activity to lipase in the presence of inhibitory substances like bile salts that bind the micelles. [Note: Colipase is secreted as the zymogen, procolipase, which is activated


Biochemistry_Lippinco

Biochemistry_Lippinco. Colipase restores activity to lipase in the presence of inhibitory substances like bile salts that bind the micelles. [Note: Colipase is secreted as the zymogen, procolipase, which is activated in the intestine by trypsin.] Orlistat, an antiobesity drug, inhibits gastric and pancreatic lipases, thereby decreasing fat absorption, resulting in weight loss.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Cholesteryl ester degradation: Most dietary cholesterol is present in the free (nonesterified) form, with 10%–15% present in the esterified form. Cholesteryl esters are hydrolyzed by pancreatic cholesteryl ester hydrolase (cholesterol esterase), which produces cholesterol plus FFA (see Fig. 15.2). Activity of this enzyme is greatly increased in the presence of bile salts. 3.


Biochemistry_Lippinco

Biochemistry_Lippinco. 3. Phospholipid degradation: Pancreatic juice is rich in the proenzyme of phospholipase A2 that, like procolipase, is activated by trypsin and, like cholesteryl ester hydrolase, requires bile salts for optimum activity. Phospholipase A2 removes one FA from carbon 2 of a phospholipid, leaving a lysophospholipid. For example, phosphatidylcholine (the predominant phospholipid of digestion) becomes lysophosphatidylcholine. The remaining FA at carbon 1 can be removed by lysophospholipase, leaving a glycerylphosphoryl base (for example, glycerylphosphorylcholine, see Fig. 15.2) that may be excreted in the feces, further degraded, or absorbed.


Biochemistry_Lippinco

Biochemistry_Lippinco. 4. Control: Pancreatic secretion of the hydrolytic enzymes that degrade dietary lipids in the small intestine is hormonally controlled (Fig. 15.4). Cells in the mucosa of the lower duodenum and jejunum produce the peptide hormone cholecystokinin (CCK), in response to the presence of lipids and partially digested proteins entering these regions of the upper small intestine. CCK acts on the gallbladder (causing it to contract and release bile, a mixture of bile salts, phospholipids, and free cholesterol) and on the exocrine cells of the pancreas (causing them to release digestive enzymes). It also decreases gastric motility, resulting in a slower release of gastric contents into the small intestine (see p. 353). Other intestinal cells produce another peptide hormone, secretin, in response to the low pH of the chyme entering the intestine from the stomach. Secretin causes the pancreas to release a solution rich in bicarbonate that helps neutralize the pH of the intestinal contents,


Biochemistry_Lippinco

Biochemistry_Lippinco. to the low pH of the chyme entering the intestine from the stomach. Secretin causes the pancreas to release a solution rich in bicarbonate that helps neutralize the pH of the intestinal contents, bringing them to the appropriate pH for digestive activity by pancreatic enzymes.


Biochemistry_Lippinco

E. Absorption by enterocytes

Biochemistry_Lippinco. E. Absorption by enterocytes


Biochemistry_Lippinco

Biochemistry_Lippinco. FFA, free cholesterol, and 2-MAG are the primary products of lipid digestion in the jejunum. These, plus bile salts and fat-soluble vitamins (A, D, E, and K), form mixed micelles (that is, disc-shaped clusters of a mixture of amphipathic lipids that coalesce with their hydrophobic groups on the inside and their hydrophilic groups on the outside). Therefore, mixed micelles are soluble in the aqueous environment of the intestinal lumen (Fig. 15.5). These particles approach the primary site of lipid absorption, the brush border membrane of the enterocytes. This microvilli-rich apical membrane is separated from the liquid contents of the intestinal lumen by an unstirred water layer that mixes poorly with the bulk fluid. The hydrophilic surface of the micelles facilitates the transport of the hydrophobic lipids through the unstirred water layer to the brush border membrane where they are absorbed. Bile salts are absorbed in the terminal ileum, with <5% being lost in the feces. [Note:


Biochemistry_Lippinco

Biochemistry_Lippinco. the hydrophobic lipids through the unstirred water layer to the brush border membrane where they are absorbed. Bile salts are absorbed in the terminal ileum, with <5% being lost in the feces. [Note: Relative to other dietary lipids, cholesterol is only poorly absorbed by the enterocytes. Drug therapy (for example, with ezetimibe) can further reduce cholesterol absorption in the small intestine.] Because short-and medium-chain FA are water soluble, they do not require the assistance of mixed micelles for absorption by the intestinal mucosa.


Biochemistry_Lippinco

Biochemistry_Lippinco. F. Triacylglycerol and cholesteryl ester resynthesis


Biochemistry_Lippinco

Biochemistry_Lippinco. The mixture of lipids absorbed by the enterocytes migrates to the smooth endoplasmic reticulum (SER) where biosynthesis of complex lipids takes place. The long-chain FA are first converted into their activated form by fatty acyl coenzyme A (CoA) synthetase (thiokinase), as shown in Figure 15.6. Using the fatty acyl CoA derivatives, the 2-MAG absorbed by the enterocytes are converted to TAG through sequential reacylations by two acyltransferases, acyl CoA:monoacylglycerol acyltransferase and acyl CoA:diacylglycerol acyltransferase. Lysophospholipids are reacylated to form phospholipids by a family of acyltransferases, and cholesterol is acylated primarily by acyl CoA:cholesterol acyltransferase (see p. 232). [Note: Virtually all long-chain FA entering the enterocytes are used in this fashion to form TAG, phospholipids, and cholesteryl esters. Short-and medium-chain FA are not converted to their CoA derivatives and are not reesterified to 2-MAG. Instead, they are released into the


Biochemistry_Lippinco

Biochemistry_Lippinco. fashion to form TAG, phospholipids, and cholesteryl esters. Short-and medium-chain FA are not converted to their CoA derivatives and are not reesterified to 2-MAG. Instead, they are released into the portal circulation, where they are carried by serum albumin to the liver.]


Biochemistry_Lippinco

Biochemistry_Lippinco. G. Lipid malabsorption Lipid malabsorption, resulting in increased lipid (including the fat-soluble vitamins and essential FA, see p. 182) in the feces, a condition known as steatorrhea, can be caused by disturbances in lipid digestion and/or absorption (Fig. 15.7). Such disturbances can result from several conditions, including CF (causing poor digestion) and short bowel syndrome (causing decreased absorption). The ability of short-and medium-chain FA to be taken up by enterocytes without the aid of mixed micelles has made them important in medical nutrition therapy for individuals with malabsorption disorders. H. Secretion from enterocytes


Biochemistry_Lippinco

Biochemistry_Lippinco. The newly resynthesized TAG and cholesteryl esters are very hydrophobic and aggregate in an aqueous environment. Therefore, they must be packaged as particles of lipid droplets surrounded by a thin layer composed of phospholipids, nonesterified cholesterol, and a molecule of the protein apolipoprotein (apo) B-48 (see p. 228). This layer stabilizes the particle and increases its solubility, thereby preventing multiple particles from coalescing. [Note: Microsomal triglyceride transfer protein is essential for the assembly of all TAG-rich apo B–containing particles in the ER (see p. 228).] The lipoprotein particles are released by exocytosis from enterocytes into the lacteals (lymphatic vessels in the villi of the small intestine). The presence of these particles in the lymph after a lipid-rich meal gives it a milky appearance. This lymph is called chyle (as opposed to chyme, the name given to the semifluid mass of partially digested food that passes from the stomach to the duodenum),


Biochemistry_Lippinco

Biochemistry_Lippinco. meal gives it a milky appearance. This lymph is called chyle (as opposed to chyme, the name given to the semifluid mass of partially digested food that passes from the stomach to the duodenum), and the particles are named chylomicrons. Chylomicrons follow the lymphatic system to the thoracic duct and are then conveyed to the left subclavian vein, where they enter the blood. The steps in the production of chylomicrons are summarized in Figure 15.6. [Note: Once released into blood, the nascent (immature) chylomicrons pick up apolipoproteins E and C-II from high-density lipoproteins and mature. (For a more detailed description of chylomicron structure and metabolism, see p. 227.)]


Biochemistry_Lippinco

Biochemistry_Lippinco. I. Use by the tissues Most of the TAG contained in chylomicrons is broken down in the capillary beds of skeletal and cardiac muscle and adipose tissue. The TAG is degraded to FFA and glycerol by lipoprotein lipase (LPL). This enzyme is synthesized and secreted primarily by adipocytes and muscle cells. Secreted LPL is anchored to the luminal surface of endothelial cells in the capillaries of muscle and adipose tissues. [Note: Familial chylomicronemia (type I hyperlipoproteinemia) is a rare, autosomal-recessive disorder caused by a deficiency of LPL or its coenzyme apo C-II (see p. 228). The result is fasting chylomicronemia and severe hypertriacylglycerolemia, which can cause pancreatitis.] 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. Fate of free fatty acids: The FFA derived from the hydrolysis of TAG may either directly enter adjacent muscle cells and adipocytes or be transported in the blood in association with serum albumin until they are taken up by cells. [Note: Human serum albumin is a large protein secreted by the liver. It transports a number of primarily hydrophobic compounds in the circulation, including FFA and some drugs.] Most cells can oxidize FA to produce energy (see p. 190). Adipocytes can also reesterify FFA to produce TAG molecules, which are stored until the FA are needed by the body (see p. 188). 2. Fate of glycerol: Glycerol released from TAG is taken up from the blood and phosphorylated by hepatic glycerol kinase to produce glycerol 3phosphate, which can enter either glycolysis or gluconeogenesis by oxidation to dihydroxyacetone phosphate (see p. 101) or be used in TAG synthesis (see p. 189). 3.


Biochemistry_Lippinco

Biochemistry_Lippinco. 3. Fate of chylomicron remnants: After most of the TAG has been removed, the chylomicron remnants (which contain cholesteryl esters, phospholipids, apolipoproteins, fat-soluble vitamins, and a small amount of TAG) bind to receptors on the liver (apo E is the ligand; see p. 229) and are endocytosed. The intracellular remnants are hydrolyzed to their component parts. Cholesterol and the nitrogenous bases of phospholipids (for example, choline) can be recycled by the body. [Note: If removal of remnants by the liver is decreased because of impaired binding to their receptor, they accumulate in the plasma. This is seen in the rare type III hyperlipoproteinemia (also called familial dysbetalipoproteinemia or broad beta disease, see p. 231).] III. CHAPTER SUMMARY


Biochemistry_Lippinco

Biochemistry_Lippinco. III. CHAPTER SUMMARY Dietary lipid digestion begins in the stomach and continues in the small intestine (Fig. 15.8). Cholesteryl esters, phospholipids, and triacylglycerols (TAG) containing long-chain-length fatty acids (FA) are degraded in the small intestine by pancreatic enzymes. The most important of these enzymes are cholesterol esterase, phospholipase A2, and pancreatic lipase.


Biochemistry_Lippinco

Biochemistry_Lippinco. In cystic fibrosis, thickened mucus prevents these enzymes reaching the intestine. In contrast, TAG in milk fat contain short-to medium-chainlength FA and are degraded in the stomach by acid lipases (lingual lipase and gastric lipase). The hydrophobic nature of lipids requires that dietary lipids be emulsified for efficient degradation. Emulsification occurs in the small intestine using peristaltic action (mechanical mixing) and bile salts (detergents). The primary products of dietary lipid degradation are 2monoacylglycerol, nonesterified (free) cholesterol, and free FA. These compounds, plus the fat-soluble vitamins, form mixed micelles that facilitate dietary lipid absorption by intestinal mucosal cells (enterocytes). These cells use activated long-chain FA to regenerate TAG and cholesteryl esters and also synthesize protein (apolipoprotein [apo] B-48), all of which are then assembled with the fat-soluble vitamins into lipoprotein particles called chylomicrons. Short-and


Biochemistry_Lippinco

Biochemistry_Lippinco. and cholesteryl esters and also synthesize protein (apolipoprotein [apo] B-48), all of which are then assembled with the fat-soluble vitamins into lipoprotein particles called chylomicrons. Short-and medium-chain FA enter blood directly. Chylomicrons are first released into the lymph and then enter the blood, where their lipid core is degraded by lipoprotein lipase (with apo C-II as the coenzyme) in the capillaries of muscle and adipose tissues. Thus, dietary lipids are made available to the peripheral tissues. Fat maldigestion or malabsorption causes steatorrhea (lipid in the feces). A deficiency in the ability to degrade chylomicron components, or remove chylomicron remnants after TAG has been degraded, results in accumulation of these particles in blood.


Biochemistry_Lippinco

Biochemistry_Lippinco. Choose the ONE best answer. 5.1. Which one of the following statements about lipid digestion is correct? A. Large lipid droplets are emulsified (have their surface area increased) in the mouth through the act of chewing (mastication). B. The enzyme colipase facilitates the binding of bile salts to mixed micelles, maximizing the activity of pancreatic lipase. C. The peptide hormone secretin causes the gallbladder to contract and release bile. D. Patients with cystic fibrosis have difficulties with digestion because their pancreatic secretions are less able to reach the small intestine, the primary site of lipid digestion. E. Formation of triacylglycerol-rich chylomicrons is independent of protein synthesis in the intestinal mucosa.


Biochemistry_Lippinco

Biochemistry_Lippinco. E. Formation of triacylglycerol-rich chylomicrons is independent of protein synthesis in the intestinal mucosa. Correct answer = D. Patients with cystic fibrosis, a genetic disease resulting in a deficiency of a functional chloride transporter, have thickened mucus that impedes the flow of pancreatic enzymes into the duodenum. Emulsification occurs through peristalsis, which provides mechanical mixing, and bile salts that function as detergents. Colipase restores activity to pancreatic lipase in the presence of inhibitory bile salts that bind the micelles. Cholecystokinin is the hormone that causes contraction of the gallbladder and release of stored bile, and secretin causes release of bicarbonate. Chylomicron formation requires synthesis of apolipoprotein B-48. 5.2. Which one of the following statements about lipid absorption from the intestine is correct? A. Dietary triacylglycerol must be completely hydrolyzed to free fatty acids and glycerol before absorption.


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Dietary triacylglycerol must be completely hydrolyzed to free fatty acids and glycerol before absorption. B. The triacylglycerol carried by chylomicrons is degraded by lipoprotein lipase, producing fatty acids that are taken up by muscle and adipose tissues and glycerol that is taken up by the liver. C. Fatty acids that contain ≤12 carbon atoms are absorbed and enter the circulation primarily via the lymphatic system. D. Deficiencies in the ability to absorb fat result in excessive amounts of chylomicrons in the blood.


Biochemistry_Lippinco

Biochemistry_Lippinco. D. Deficiencies in the ability to absorb fat result in excessive amounts of chylomicrons in the blood. Correct answer = B. The triacylglycerols (TAG) in chylomicrons are degraded to fatty acids (FA) and glycerol by lipoprotein lipase on capillary endothelial surfaces in muscle and adipose tissue, thus providing a source of FA to these tissues for degradation or storage and providing glycerol for hepatic metabolism. In the duodenum, TAG are degraded to one 2-monoacylglycerol + two free FA that get absorbed. Medium-and short-chain FA enter directly into blood (not lymph), and they neither require micelles nor get packaged into chylomicrons. Because chylomicrons contain dietary lipids that were digested and absorbed, a defect in fat absorption would result in decreased production of chylomicrons. Fatty Acid, Triacylglycerol, and Ketone Body Metabolism 16 For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW


Biochemistry_Lippinco

Biochemistry_Lippinco. Fatty acids exist free in the body (that is, they are nonesterified) and as fatty acyl esters in more complex molecules such as triacylglycerols (TAG). Low levels of free fatty acids (FFA) occur in all tissues, but substantial amounts can sometimes be found in the plasma, particularly during fasting. Plasma FFA (transported on serum albumin) are in route from their point of origin (TAG of adipose tissue or circulating lipoproteins) to their site of consumption (most tissues). FFA can be oxidized by many tissues, particularly liver and muscle, to provide energy and, in the liver, to provide the substrate for ketone body synthesis. Fatty acids are also structural components of membrane lipids, such as phospholipids and glycolipids (see p. 201). Fatty acids attached to certain proteins enhance the ability of those proteins to associate with membranes (see p. 206). Fatty acids are also precursors of the hormone-like prostaglandins (see p. 213). Esterified fatty acids, in the form of TAG


Biochemistry_Lippinco

Biochemistry_Lippinco. the ability of those proteins to associate with membranes (see p. 206). Fatty acids are also precursors of the hormone-like prostaglandins (see p. 213). Esterified fatty acids, in the form of TAG stored in white adipose tissue (WAT), serve as the major energy reserve of the body. Alterations in fatty acid metabolism are associated with obesity and diabetes. Figure 16.1 illustrates the metabolic pathways of fatty acid synthesis and degradation and their relationship to carbohydrate metabolism.


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Biochemistry_Lippinco. II. FATTY ACID STRUCTURE A fatty acid consists of a hydrophobic hydrocarbon chain with a terminal carboxyl group that has a pKa (see p. 6) of ~4.8 (Fig. 16.2). At physiologic pH, the terminal carboxyl group (–COOH) ionizes, becoming –COO−. [Note: When the pH is above the pK, the deprotonated form predominates (see p. 7).] This anionic group has an affinity for water, giving the fatty acid its amphipathic nature (having both a hydrophilic and a hydrophobic region). However, for longchain-length fatty acids (LCFA), the hydrophobic portion is predominant. These molecules are highly water insoluble and must be transported in the circulation in association with protein. More than 90% of the fatty acids found in plasma are in the form of fatty acid esters (primarily TAG, cholesteryl esters, and phospholipids) contained in circulating lipoprotein particles (see p. 227). FFA are transported in the circulation in association with albumin, the most abundant protein in serum.


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Fatty acid saturation Fatty acid chains may contain no double bonds (that is, be saturated) or contain one or more double bonds (that is, be mono-or polyunsaturated). In humans, the majority are saturated or monounsaturated. When double bonds are present, they are nearly always in the cis rather than in the trans configuration. The introduction of a cis double bond causes the fatty acid to bend or kink at that position (Fig. 16.3). If the fatty acid has two or more double bonds, they are always spaced at three-carbon intervals. [Note: In general, addition of double bonds decreases the melting temperature (Tm) of a fatty acid, whereas increasing the chain length increases the Tm. Because membrane lipids typically contain LCFA, the presence of double bonds in some fatty acids helps maintain the fluid nature of those lipids. See p. 363 for a discussion of the dietary occurrence of cis and trans unsaturated fatty acids.] B. Fatty acid chain length and double bond positions


Biochemistry_Lippinco

Biochemistry_Lippinco. The common names and structures of some fatty acids of physiologic importance are listed in Figure 16.4. In humans, fatty acids with an even number of carbon atoms (16, 18, or 20) predominate, with longer fatty acids (>22 carbons) being found in the brain. The carbon atoms are numbered, beginning with the carbonyl carbon as carbon 1. The number before the colon indicates the number of carbons in the chain, and those after the colon indicate the numbers and positions (relative to the carboxyl end) of double bonds. For example, as denoted in Figure 16.4, arachidonic acid, 20:4(5,8,11,14), is 20 carbons long and has four double bonds (between carbons 5–6, 8–9, 11–12, and 14–15). [Note: Carbon 2, the carbon to which the carboxyl group is attached, is also called the α-carbon, carbon 3 is the βcarbon, and carbon 4 is the γ-carbon. The carbon of the terminal methyl group is called the ω-carbon regardless of the chain length.] The double bonds in a fatty acid can also be referenced relative


Biochemistry_Lippinco

Biochemistry_Lippinco. βcarbon, and carbon 4 is the γ-carbon. The carbon of the terminal methyl group is called the ω-carbon regardless of the chain length.] The double bonds in a fatty acid can also be referenced relative to the ω (methyl) end of the chain. Arachidonic acid is referred to as an ω-6 fatty acid because the terminal double bond is six bonds from the ω end (Fig. 16.5A). [Note: The equivalent designation of n-6 may also be used (Fig. 16.5B).] Another ω-6 fatty acid is the essential linoleic acid 18:2(9,12). In contrast, α-linolenic acid, 18:3(9,12,15), is an essential ω-3 fatty acid.


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Biochemistry_Lippinco. C. Essential fatty acids Linoleic acid, the precursor of ω-6 arachidonic acid that is the substrate for prostaglandin synthesis (see p. 213), and α-linolenic acid, the precursor of ω-3 fatty acids that are important for growth and development, are dietary essentials in humans because we lack the enzymes needed to synthesize them. Plants provide us with these essential fatty acids. [Note: Arachidonic acid becomes essential if linoleic acid is deficient in the diet. See p. 362 for a discussion of the nutritional significance of ω-3 and ω-6 fatty acids.] Essential fatty acid deficiency (rare) can result in a dry, scaly dermatitis as a result of an inability to synthesize molecules that provide the water barrier in skin (see p. 206). III. FATTY ACID DE NOVO SYNTHESIS