Biochemistry_Lippinco

Biochemistry_Lippinco. 80) and acyl CoA dehydrogenases (see p. 192). CoQ transfers electrons to Complex III (cytochrome bc1). Thus, a function of CoQ is to link the flavoprotein dehydrogenases to the cytochromes. 5. Cytochromes: The remaining members of the ETC are cytochrome proteins. Each contains a heme group (a porphyrin ring plus iron). Unlike the heme groups of hemoglobin, the cytochrome iron is reversibly converted from its ferric (Fe3+) to its ferrous (Fe2+) form as a normal part of its function as an acceptor and donor of electrons. Electrons are passed along the chain from cytochrome bc1 (Complex III), to cytochrome c, and then to cytochromes a + a3 ([Complex IV] see Fig. 6.8). As electrons flow, four H+ are pumped across the inner mitochondrial membrane at Complex III and two at Complex IV. [Note: Cytochrome c is located in the intermembrane space, loosely associated with the outer face of the inner membrane. As seen with CoQ, cytochrome c is a mobile electron carrier.] 6.


Biochemistry_Lippinco

Biochemistry_Lippinco. Cytochrome a + a3: Because this cytochrome complex (Complex IV) is the only electron carrier in which the heme iron has an available coordination site that can react directly with O2, it also is called cytochrome c oxidase. At Complex IV, the transported electrons, O2, and free H+ are brought together, and O2 is reduced to H2O (see Fig. 6.8). [Note: Four electrons are required to reduce one molecule of O2 to two molecules of H2O.] Cytochrome c oxidase contains Cu atoms that are required for this complicated reaction to occur. Electrons move from CuA to cytochrome a to cytochrome a3 (in association with CuB) to O2. 7.


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Biochemistry_Lippinco. 7. Site-specific inhibitors: Inhibitors of specific sites in the ETC have been identified and are illustrated in Figure 6.10. These respiratory inhibitors prevent the passage of electrons by binding to a component of the chain, blocking the oxidation-reduction reaction. Therefore, all electron carriers before the block are fully reduced, whereas those located after the block are oxidized. [Note: Inhibition of the ETC inhibits ATP synthesis because these processes are tightly coupled (see p. 78).] NaN3 = sodium azide. Leakage of electrons from the ETC produces reactive oxygen species (ROS), such as superoxide (O2−·), hydrogen peroxide (H2O2), and hydroxyl radicals (OH·). ROS damage DNA and proteins and cause lipid peroxidation. Enzymes such as superoxide dismutase (SOD), catalase, and glutathione peroxidase are cellular defenses against ROS (see p. 148). D. Free energy release during electron transport


Biochemistry_Lippinco

Biochemistry_Lippinco. D. Free energy release during electron transport The free energy released as electrons are transferred along the ETC from an electron donor (reducing agent or reductant) to an electron acceptor (oxidizing agent or oxidant) is used to pump H+ at Complexes I, III, and IV. [Note: The electrons can be transferred as hydride ions to NAD+; as hydrogen atoms to FMN, CoQ, and FAD; or as electrons to cytochromes.] 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. Redox pairs: Oxidation (loss of electrons) of one substance is always accompanied by reduction (gain of electrons) of a second. For example, Figure 6.11 shows the oxidation of NADH to NAD+ by NADH dehydrogenase at Complex I, accompanied by the reduction of FMN, the prosthetic group, to FMNH2. Such redox reactions can be written as the sum of two separate half reactions, one an oxidation and the other a reduction (see Fig. 6.11). NAD+ and NADH form a redox pair, as do FMN and FMNH2. Redox pairs differ in their tendency to lose electrons. This tendency is a characteristic of a particular redox pair and can be quantitatively specified by a constant, E0 (the standard reduction potential), with units in volts. 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Standard reduction potential: The E0 of various redox pairs can be ordered from the most negative E0 to the most positive. The more negative the E0 of a redox pair, the greater the tendency of the reductant member of that pair to lose electrons. The more positive the E0, the greater the tendency of the oxidant member of that pair to accept electrons. Therefore, electrons flow from the pair with the more negative E0 to that with the more positive E0. The E0 values for some members of the ETC are shown in Figure 6.12. [Note: The components of the chain are arranged in order of increasingly positive E0 values.] 3. Relationship of ∆G0 to ∆E0: The ∆G0 is related directly to the magnitude of the change in E0: where n = number of electrons transferred (1 for a cytochrome, 2 for NADH, FADH2, and CoQ)


Biochemistry_Lippinco

Biochemistry_Lippinco. F = Faraday constant (23.1 kcal/volt mol) ∆E0 = E0 of the electron-accepting pair minus the E0 of the electron-donating pair ∆G0 = change in the standard free energy 4. ∆G0 of ATP: The ∆G0 for the phosphorylation of ADP to ATP is +7.3 kcal/mol. The transport of a pair of electrons from NADH to O2 through the ETC releases 52.6 kcal. Therefore, more than sufficient energy is available to produce three ATP from three ADP and three Pi (3 × 7.3 = 21.9 kcal/mol), sometimes expressed as a P/O ratio (ATP made per O atom reduced) of 3:1. The remaining calories are used for ancillary reactions or released as heat. [Note: The P:O for FADH2 is 2:1 because Complex I is bypassed.] VI. PHOSPHORYLATION OF ADP TO ATP The transfer of electrons down the ETC is energetically favored because NADH is a strong electron donor and O2 is an avid electron acceptor. However, the flow of electrons does not directly result in ATP synthesis. A. Chemiosmotic hypothesis


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Chemiosmotic hypothesis The chemiosmotic hypothesis (also known as the Mitchell hypothesis) explains how the free energy generated by the transport of electrons by the ETC is used to produce ATP from ADP + Pi. 1. Proton pump: Electron transport is coupled to ADP phosphorylation by the pumping of H+ across the inner mitochondrial membrane, from the matrix to the intermembrane space, at Complexes I, III, and IV. For each pair of electrons transferred from NADH to O2, 10 H+ are pumped. This creates an electrical gradient (with more positive charges on the cytosolic side of the membrane than on the matrix side) and a pH (chemical) gradient (the cytosolic side of the membrane is at a lower pH than the matrix side), as shown in Figure 6.13. The energy (proton-motive force) generated by these gradients is sufficient to drive ATP synthesis. Thus, the H+ gradient serves as the common intermediate that couples oxidation to phosphorylation. 2.


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Biochemistry_Lippinco. ATP synthase: The multisubunit enzyme ATP synthase ([Complex V] Fig. 6.14) synthesizes ATP using the energy of the H+ gradient. It contains a membrane domain (Fo) that spans the inner mitochondrial membrane and an extramembranous domain (F1) that appears as a sphere that protrudes into the mitochondrial matrix (see Fig. 6.13). The chemiosmotic hypothesis proposes that after H+ have been pumped to the cytosolic side of the inner mitochondrial membrane, they reenter the matrix by passing through a H+ channel in the Fo domain, driving rotation of the c ring of Fo and, at the same time, dissipating the pH and electrical gradients. Rotation in Fo causes conformational changes in the three β subunits of F1 that allow them to bind ADP + Pi, phosphorylate ADP to ATP, and release ATP. One complete rotation of the c ring produces three ATP. [Note: ATP synthase is also called F1/Fo-ATPase because the enzyme can also catalyze the hydrolysis of ATP to ADP and Pi.] contains eight subunits. One


Biochemistry_Lippinco

Biochemistry_Lippinco. rotation of the c ring produces three ATP. [Note: ATP synthase is also called F1/Fo-ATPase because the enzyme can also catalyze the hydrolysis of ATP to ADP and Pi.] contains eight subunits. One complete turn of the ring is driven by eight H+ (protons) moving through the Fo domain. The resulting conformational changes in the three β subunits of the F1 domain allow phosphorylation of three adenosine diphosphates (ADP) to three ATP.] Pi = inorganic phosphate.


Biochemistry_Lippinco

Biochemistry_Lippinco. a. Coupling in oxidative phosphorylation: In normal mitochondria, ATP synthesis is coupled to electron transport through the H+ gradient. Increasing (or decreasing) one process has the same effect on the other. For example, hydrolysis of ATP to ADP and Pi in energy-requiring reactions increases the availability of substrates for ATP synthase and, thus, increases H+ flow through the enzyme. Electron transport and H+ pumping by the ETC increase to maintain the H+ gradient and allow ATP synthesis.


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Biochemistry_Lippinco. b. Oligomycin: This drug binds to the Fo (hence the letter “o”) domain of ATP synthase, closing the H+ channel and preventing reentry of H+ into the matrix, thereby inhibiting phosphorylation of ADP to ATP. Because the pH and electrical gradients cannot be dissipated in the presence of this phosphorylation inhibitor, electron transport stops because of the difficulty of pumping any more H+ against the steep gradient. This dependency of cellular respiration on the ability to phosphorylate ADP to ATP is known as respiratory control and is the consequence of the tight coupling of these processes. c.


Biochemistry_Lippinco

Biochemistry_Lippinco. c. Uncoupling proteins: Uncoupling proteins (UCP) occur in the inner mitochondrial membrane of mammals, including humans. These proteins form channels that allow H+ to reenter the mitochondrial matrix without energy being captured as ATP (Fig. 6.15). The energy is released as heat, and the process is called nonshivering thermogenesis. UCP1, also called thermogenin, is responsible for heat production in the mitochondria-rich brown adipocytes of mammals. [Note: Cold causes catecholamine-dependent activation of UCP1 expression.] In brown fat, unlike the more abundant white fat, ~90% of its respiratory energy is used for thermogenesis in infants in response to cold. Thus, brown fat is involved in energy expenditure, whereas white fat is involved in energy storage. [Note: Brown fat depots have recently been shown to be present in adults.] d.


Biochemistry_Lippinco

Biochemistry_Lippinco. Synthetic uncouplers: Electron transport and phosphorylation of ADP can also be uncoupled by compounds that shuttle H+ across the inner mitochondrial membrane, dissipating the gradient. The classic example is 2,4-dinitrophenol, a lipophilic H+ carrier (ionophore) that readily diffuses through the mitochondrial membrane (Fig. 6.16). This uncoupler causes electron transport to proceed at a rapid rate without establishing a H+ gradient, much as do the UCP. Again, energy is released as heat rather than being used to synthesize ATP. [Note: In high doses, aspirin and other salicylates uncouple oxidative phosphorylation. This explains the fever that accompanies toxic overdoses of these drugs.] B. Membrane transport systems The inner mitochondrial membrane is impermeable to most charged or hydrophilic substances. However, it contains numerous transport proteins that permit passage of certain molecules from the cytosol to the mitochondrial matrix. 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. ATP and ADP transport: The inner membrane requires specialized carriers to transport ADP and Pi from the cytosol (where ATP is hydrolyzed to ADP in many energy-requiring reactions) into mitochondria, where ATP can be resynthesized. An adenine nucleotide antiporter imports one ADP from the cytosol into the matrix, while exporting one ATP from the matrix into the cytosol (see Fig. 6.13). A symporter cotransports Pi and H+ from the cytosol into the matrix. 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. Reducing equivalent transport: The inner mitochondrial membrane lacks an NADH transporter, and NADH produced in the cytosol (for example, in glycolysis; see p. 101) cannot directly enter the mitochondrial matrix. However, reducing equivalents of NADH are transported from the cytosol into the matrix using substrate shuttles. In the glycerol 3phosphate shuttle (Fig. 6.17A), two electrons are transferred from NADH to dihydroxyacetone phosphate by cytosolic glycerol 3-phosphate dehydrogenase. The glycerol 3-phosphate produced is oxidized by the mitochondrial isozyme as FAD is reduced to FADH2. CoQ of the ETC oxidizes the FADH2. Therefore, the glycerol 3-phosphate shuttle results in the synthesis of two ATP for each cytosolic NADH oxidized. This contrasts with the malate-aspartate shuttle (Fig. 6.17B), which produces NADH (rather than FADH2) in the mitochondrial matrix, thereby yielding three ATP for each cytosolic NADH oxidized by malate dehydrogenase as oxaloacetate is reduced to malate.


Biochemistry_Lippinco

Biochemistry_Lippinco. 6.17B), which produces NADH (rather than FADH2) in the mitochondrial matrix, thereby yielding three ATP for each cytosolic NADH oxidized by malate dehydrogenase as oxaloacetate is reduced to malate. A transport protein moves malate into the mitochondrial matrix.


Biochemistry_Lippinco

Biochemistry_Lippinco. inner mitochondrial membrane. A. Glycerol 3-phosphate shuttle. B. Malate aspartate shuttle. DHAP = dihydroxyacetone phosphate; NAD(H) = dinucleotide; CoQ = coenzyme Q. C. Inherited defects in oxidative phosphorylation


Biochemistry_Lippinco

Biochemistry_Lippinco. Thirteen of the ~90 polypeptides required for oxidative phosphorylation are encoded by mtDNA and synthesized in mitochondria, whereas the remaining proteins are encoded by nuclear DNA, synthesized in the cytosol, and then transported into mitochondria. Defects in oxidative phosphorylation are more likely a result of alterations in mtDNA, which has a mutation rate about 10 times greater than that of nuclear DNA. Tissues with the greatest ATP requirement (for example, the central nervous system, skeletal and heart muscle, and the liver) are most affected by defects in oxidative phosphorylation. Mutations in mtDNA are responsible for several diseases, including some cases of mitochondrial myopathies, and Leber hereditary optic neuropathy, a disease in which bilateral loss of central vision occurs as a result of neuroretinal degeneration, including damage to the optic nerve. [Note: mtDNA is maternally inherited because mitochondria from the sperm cell do not enter the fertilized egg.]


Biochemistry_Lippinco

Biochemistry_Lippinco. D. Mitochondria and apoptosis The process of apoptosis (programmed cell death) may be initiated through the intrinsic (mitochondrial-mediated) pathway by the formation of pores in the outer mitochondrial membrane. These pores allow cytochrome c to leave the intermembrane space and enter the cytosol. There, cytochrome c, in association with proapoptotic factors, activates a family of proteolytic enzymes (the caspases), causing cleavage of key proteins and resulting in the morphologic and biochemical changes characteristic of apoptosis. VII. CHAPTER SUMMARY


Biochemistry_Lippinco

Biochemistry_Lippinco. The change in free energy (∆G) occurring during a reaction predicts the direction in which that reaction will spontaneously proceed. If ∆G is negative (that is, the product has a lower free energy than the substrate), then the reaction is spontaneous as written. If ∆G is positive, then the reaction is not spontaneous. If ∆G = 0, then the reaction is in equilibrium. The ∆G of the forward reaction is equal in magnitude but opposite in sign to that of the back reaction. The ∆G are additive in any sequence of consecutive reactions, as are the standard free energy changes (∆G0). Therefore, reactions or processes that have a large, positive ∆G are made possible by coupling with those that have a large, negative ∆G such as ATP hydrolysis. The reduced coenzymes nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2) each donate a pair of electrons to a specialized set of electron carriers, consisting of flavin mononucleotide (FMN), iron-sulfur centers, coenzyme Q, and


Biochemistry_Lippinco

Biochemistry_Lippinco. and flavin adenine dinucleotide (FADH2) each donate a pair of electrons to a specialized set of electron carriers, consisting of flavin mononucleotide (FMN), iron-sulfur centers, coenzyme Q, and a series of heme-containing cytochromes, collectively called the electron transport chain. This pathway is present in the inner mitochondrial membrane (impermeable to most substances) and is the final common pathway by which electrons derived from different fuels of the body flow to oxygen (O2), which has a large, positive reduction potential (E0), reducing it to water. The terminal cytochrome, cytochrome c oxidase, is the only cytochrome able to bind O2. Electron transport results in the pumping of protons (H+) across the inner mitochondrial membrane from the matrix to the intermembrane space, 10 H+ per NADH oxidized. This process creates electrical and pH gradients across the inner mitochondrial membrane. After H+ have been transferred to the cytosolic side of the membrane, they reenter the


Biochemistry_Lippinco

Biochemistry_Lippinco. H+ per NADH oxidized. This process creates electrical and pH gradients across the inner mitochondrial membrane. After H+ have been transferred to the cytosolic side of the membrane, they reenter the matrix by passing through the Fo H+ channel in ATP synthase (Complex V), dissipating the pH and electrical gradients and causing conformational changes in the F1 β subunits of the synthase that result in the synthesis of ATP from ADP + inorganic phosphate. Electron transport and phosphorylation are tightly coupled in oxidative phosphorylation ([OXPHOS] Fig. 6.18). Inhibition of one process inhibits the other. These processes can be uncoupled by uncoupling protein-1 of the inner mitochondrial membrane of brown adipocytes and by synthetic compounds such as 2,4-dinitrophenol and aspirin, all of which dissipate the H+ gradient. In uncoupled mitochondria, the energy produced by electron transport is released as heat rather than being used to synthesize ATP. Mutations in mitochondrial DNA, which


Biochemistry_Lippinco

Biochemistry_Lippinco. dissipate the H+ gradient. In uncoupled mitochondria, the energy produced by electron transport is released as heat rather than being used to synthesize ATP. Mutations in mitochondrial DNA, which is maternally inherited, are responsible for some cases of mitochondrial diseases such as Leber hereditary optic neuropathy. The release of cytochrome c into the cytoplasm and subsequent activation of proteolytic caspases results in apoptotic cell death.


Biochemistry_Lippinco

Biochemistry_Lippinco. Choose the ONE best answer. .1. 2,4-Dinitrophenol (DNP), an uncoupler of oxidative phosphorylation, was used as a weight-loss agent in the 1930s. Reports of fatal overdoses led to its discontinuation in 1939. Which of the following would most likely be true concerning individuals taking 2,4-DNP? A. ATP levels in the mitochondria are greater than normal. B. Body temperature is elevated as a result of hypermetabolism. C. Cyanide has no effect on electron flow. D. The proton gradient across the inner mitochondrial membrane is greater than normal. E. The rate of electron transport is abnormally low.


Biochemistry_Lippinco

Biochemistry_Lippinco. C. Cyanide has no effect on electron flow. D. The proton gradient across the inner mitochondrial membrane is greater than normal. E. The rate of electron transport is abnormally low. Correct answer = B. When phosphorylation is uncoupled from electron flow, a decrease in the proton gradient across the inner mitochondrial membrane and, therefore, impaired ATP synthesis are expected. In an attempt to compensate for this defect in energy capture, metabolism and electron flow to oxygen are increased. This hypermetabolism will be accompanied by elevated body temperature because the energy in fuels is largely wasted, appearing as heat. The electron transport chain will still be inhibited by cyanide. .2. Which of the following has the strongest tendency to gain electrons? A. Coenzyme Q B. Cytochrome c C. Flavin adenine dinucleotide D. Nicotinamide adenine dinucleotide E. Oxygen


Biochemistry_Lippinco

Biochemistry_Lippinco. .2. Which of the following has the strongest tendency to gain electrons? A. Coenzyme Q B. Cytochrome c C. Flavin adenine dinucleotide D. Nicotinamide adenine dinucleotide E. Oxygen Correct answer = E. Oxygen is the terminal acceptor of electrons in the electron transport chain (ETC). Electrons flow down the ETC to oxygen because it has the highest (most positive) reduction potential (E0). The other choices precede oxygen in the ETC and have lower E0 values. .3. Explain why and how the malate-aspartate shuttle moves nicotinamide adenine dinucleotide reducing equivalents from the cytosol to the mitochondrial matrix.


Biochemistry_Lippinco

Biochemistry_Lippinco. .3. Explain why and how the malate-aspartate shuttle moves nicotinamide adenine dinucleotide reducing equivalents from the cytosol to the mitochondrial matrix. There is no transporter for nicotinamide adenine dinucleotide (NADH) in the inner mitochondrial membrane. However, cytoplasmic NADH can be oxidized to NAD+ by malate dehydrogenase as oxaloacetate (OAA) is reduced to malate. The malate is transported across the inner membrane to the matrix where the mitochondrial isozyme of malate dehydrogenase oxidizes it to OAA as mitochondrial NAD+ is reduced to NADH. This NADH can be oxidized by Complex I of the electron transport chain, generating three ATP through the coupled processes of oxidative phosphorylation. .4. Carbon monoxide (CO) binds to and inhibits Complex IV of the electron transport chain. What effect, if any, should this respiratory inhibitor have on phosphorylation of adenosine diphosphate (ADP) to ATP?


Biochemistry_Lippinco

Biochemistry_Lippinco. Inhibition of electron transport by respiratory inhibitors such as CO results in an inability to maintain the proton (H+) gradient. Therefore, phosphorylation of ADP to ATP is inhibited, as are ancillary reactions such as calcium uptake by mitochondria, because they also require the H+ gradient. Introduction to Carbohydrates 7 For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW


Biochemistry_Lippinco

Biochemistry_Lippinco. Introduction to Carbohydrates 7 For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW Carbohydrates (saccharides) are the most abundant organic molecules in nature. They have a wide range of functions, including providing a significant fraction of the dietary calories for most organisms, acting as a storage form of energy in the body, and serving as cell membrane components that mediate some forms of intercellular communication. Carbohydrates also serve as a structural component of many organisms, including the cell walls of bacteria, the exoskeleton of insects, and the fibrous cellulose of plants. [Note: The full set of carbohydrates produced by an organism is its glycome.] The empiric formula for many of the simpler carbohydrates is (CH2O)n, where n ≥3, hence the name “hydrate of carbon.” II. CLASSIFICATION AND STRUCTURE


Biochemistry_Lippinco

Biochemistry_Lippinco. Monosaccharides (simple sugars) can be classified according to the number of carbon atoms they contain. Examples of some monosaccharides commonly found in humans are listed in Figure 7.1. They can also be classified by the type of carbonyl group they contain. Carbohydrates with an aldehyde as their carbonyl group are called aldoses, whereas those with a keto as their carbonyl group are called ketoses (Fig. 7.2). For example, glyceraldehyde is an aldose, whereas dihydroxyacetone is a ketose. Carbohydrates that have a free carbonyl group have the suffix -ose. [Note: Ketoses have an additional “ul” in their suffix such as xylulose. There are exceptions, such as fructose, to this rule.] Monosaccharides can be linked by glycosidic bonds to create larger structures (Fig. 7.3). Disaccharides contain two monosaccharide units, oligosaccharides contain three to ten monosaccharide units, and polysaccharides contain more than ten monosaccharide units and can be hundreds of sugar units in length.


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Isomers and epimers Compounds that have the same chemical formula but have different structures are called isomers. For example, fructose, glucose, mannose, and galactose are all isomers of each other, having the same chemical formula, C6H12O6. Carbohydrate isomers that differ in configuration around only one specific carbon atom (with the exception of the carbonyl carbon, see C. 1. below) are defined as epimers of each other. For example, glucose and galactose are C-4 epimers because their structures differ only in the position of the –OH (hydroxyl) group at carbon 4. [Note: The carbons in sugars are numbered beginning at the end that contains the carbonyl carbon (that is, the aldehyde or keto group), as shown in Fig. 7.4.] Glucose and mannose are C-2 epimers. However, because galactose and mannose differ in the position of –OH groups at two carbons (carbons 2 and 4), they are isomers rather than epimers (see Fig. 7.4). B. Enantiomers


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Enantiomers A special type of isomerism is found in the pairs of structures that are mirror images of each other. These mirror images are called enantiomers, and the two members of the pair are designated as a D-and an L-sugar (Fig. 7.5). The vast majority of the sugars in humans are D-isomers. In the D-isomeric form, the –OH group on the asymmetric carbon (a carbon linked to four different atoms or groups) farthest from the carbonyl carbon is on the right, whereas in the L-isomer, it is on the left. Most enzymes are specific for either the D or the L form, but enzymes known as isomerases are able to interconvert D-and L-isomers. comparison to a triose, glyceraldehyde. [Note: The asymmetric carbons are shown in green.] C. Monosaccharide cyclization


Biochemistry_Lippinco

Biochemistry_Lippinco. comparison to a triose, glyceraldehyde. [Note: The asymmetric carbons are shown in green.] C. Monosaccharide cyclization Less than 1% of each of the monosaccharides with five or more carbons exists in the open-chain (acyclic) form in solution. Rather, they are predominantly found in a ring (cyclic) form, in which the aldehyde (or keto) group has reacted with a hydroxyl group on the same sugar, making the carbonyl carbon (carbon 1 for an aldose, carbon 2 for a ketose) asymmetric. This asymmetric carbon is referred to as the anomeric carbon.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. Anomers: Creation of an anomeric carbon (the former carbonyl carbon) generates a new pair of isomers, the α and β configurations of the sugar (for example, α-D-glucopyranose and β-D-glucopyranose), as shown in Figure 7.6, that are anomers of each other. [Note: In the α configuration, the –OH group on the anomeric carbon projects to the same side as the ring in a modified Fischer projection formula (see Fig. 7.6A) and is trans to the CH2OH group in a Haworth projection formula (see Fig. 7.6B).


Biochemistry_Lippinco

Biochemistry_Lippinco. The α and β forms are not mirror images, and they are referred to as diastereomers.] Enzymes are able to distinguish between these two structures and use one or the other preferentially. For example, glycogen is synthesized from α-D-glucopyranose, whereas cellulose is synthesized from β-D-glucopyranose. The cyclic α and β anomers of a sugar in solution spontaneously (but slowly) form an equilibrium mixture, a process known as mutarotation (see Fig. 7.6). [Note: For glucose, the α form makes up 36% of the mixture.] six-membered ring (5 C + 1 O) is termed a pyranose, whereas one with a five-membered ring (4 C + 1 O) is a furanose. Virtually all glucose in solution is in the pyranose form.] 2. Reducing sugars: If the hydroxyl group on the anomeric carbon of a cyclized sugar is not linked to another compound by a glycosidic bond (see E. below), the ring can open. The sugar can act as a reducing agent and is termed a reducing sugar. Such sugars can react with chromogenic agents (for


Biochemistry_Lippinco

Biochemistry_Lippinco. to another compound by a glycosidic bond (see E. below), the ring can open. The sugar can act as a reducing agent and is termed a reducing sugar. Such sugars can react with chromogenic agents (for example, the Benedict reagent) causing the reagent to be reduced and colored as the aldehyde group of the acyclic sugar is oxidized to a carboxyl group. All monosaccharides, but not all disaccharides, are reducing sugars. [Note: Fructose, a ketose, is a reducing sugar because it can be isomerized to an aldose.]


Biochemistry_Lippinco

Biochemistry_Lippinco. A colorimetric test can detect a reducing sugar in urine. A positive result is indicative of an underlying pathology (because sugars are not normally present in urine) and can be followed up by more specific tests to identify the reducing sugar. D. Monosaccharide joining Monosaccharides can be joined to form disaccharides, oligosaccharides, and polysaccharides. Important disaccharides include lactose (galactose + glucose), sucrose (glucose + fructose), and maltose (glucose + glucose). Important polysaccharides include branched glycogen (from animal sources) and starch (plant sources) and unbranched cellulose (plant sources). Each is a polymer of glucose. E. Glycosidic bonds


Biochemistry_Lippinco

Biochemistry_Lippinco. E. Glycosidic bonds The bonds that link sugars are called glycosidic bonds. They are formed by enzymes known as glycosyltransferases that use nucleotide sugars (activated sugars) such as uridine diphosphate glucose as substrates. Glycosidic bonds between sugars are named according to the numbers of the connected carbons and with regard to the position of the anomeric hydroxyl group of the first sugar involved in the bond. If this anomeric hydroxyl is in the α configuration, then the linkage is an α-bond. If it is in the β configuration, then the linkage is a β-bond. Lactose, for example, is synthesized by forming a glycosidic bond between carbon 1 of β-galactose and carbon 4 of glucose. Therefore, the linkage is a β(1→4) glycosidic bond (see Fig. 7.3). [Note: Because the anomeric end of the glucose residue is not involved in the glycosidic linkage, it (and, therefore, lactose) remains a reducing sugar.] F. Carbohydrate linkage to noncarbohydrates


Biochemistry_Lippinco

Biochemistry_Lippinco. F. Carbohydrate linkage to noncarbohydrates Carbohydrates can be attached by glycosidic bonds to noncarbohydrate structures, including purine and pyrimidine bases (found in nucleic acids), aromatic rings (such as those found in steroids and bilirubin), proteins (found in glycoproteins and proteoglycans), and lipids (found in glycolipids). If the group on the noncarbohydrate molecule to which the sugar is attached is an –NH2 group, then the bond is called an N-glycosidic link. If the group is an –OH, then the bond is an O-glycosidic link (Fig. 7.7). [Note: All sugar-sugar glycosidic bonds are O-type linkages.] III. DIETARY CARBOHYDRATE DIGESTION


Biochemistry_Lippinco

Biochemistry_Lippinco. III. DIETARY CARBOHYDRATE DIGESTION The principal sites of dietary carbohydrate digestion are the mouth and intestinal lumen. This digestion is rapid and is catalyzed by enzymes known as glycoside hydrolases (glycosidases) that hydrolyze glycosidic bonds (Fig. 7.8). Because little monosaccharide is present in diets of mixed animal and plant origin, the enzymes are primarily endoglycosidases that hydrolyze polysaccharides and oligosaccharides and disaccharidases that hydrolyze tri-and disaccharides into their reducing sugar components. Glycosidases are usually specific for the structure and configuration of the glycosyl residue to be removed as well as for the type of bond to be broken. The final products of carbohydrate digestion are the monosaccharides glucose, galactose, and fructose that are absorbed by cells (enterocytes) of the small intestine. A. Salivary α-amylase


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Salivary α-amylase The major dietary polysaccharides are of plant (starch, composed of amylose and amylopectin) and animal (glycogen) origin. During mastication (chewing), salivary α-amylase acts briefly on dietary starch and glycogen, hydrolyzing random α(1→4) bonds. [Note: There are both α(1→4)-and β(1→4)-endoglucosidases in nature, but humans do not produce the latter. Therefore, we are unable to digest cellulose, a carbohydrate of plant origin containing β(1→4) glycosidic bonds between glucose residues.] Because branched amylopectin and glycogen also contain α(1→6) bonds, which α-amylase cannot hydrolyze, the digest resulting from its action contains a mixture of short, branched and unbranched oligosaccharides known as dextrins (Fig. 7.9). [Note: Disaccharides are also present as they, too, are resistant to amylase.] Carbohydrate digestion halts temporarily in the stomach, because the high acidity inactivates salivary α-amylase. B. Pancreatic α-amylase


Biochemistry_Lippinco

Biochemistry_Lippinco. Carbohydrate digestion halts temporarily in the stomach, because the high acidity inactivates salivary α-amylase. B. Pancreatic α-amylase When the acidic stomach contents reach the small intestine, they are neutralized by bicarbonate secreted by the pancreas, and pancreatic αamylase continues the process of starch digestion. C. Intestinal disaccharidases


Biochemistry_Lippinco

Biochemistry_Lippinco. C. Intestinal disaccharidases The final digestive processes occur primarily at the mucosal lining of the duodenum and upper jejunum and include the action of several disaccharidases (see Fig. 7.9). For example, isomaltase cleaves the α(1→6) bond in isomaltose, and maltase cleaves the α(1→4) bond in maltose and maltotriose, each producing glucose. Sucrase cleaves the α(1→2) bond in sucrose, producing glucose and fructose, and lactase (β-galactosidase) cleaves the β(1→4) bond in lactose, producing galactose and glucose. [Note: The substrates for isomaltase are broader than its name suggests, and it hydrolyzes the majority of maltose.] Trehalose, an α(1→1) disaccharide of glucose found in mushrooms and other fungi, is cleaved by trehalase. These enzymes are transmembrane proteins of the brush border on the luminal (apical) surface of the enterocytes.


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Biochemistry_Lippinco. Sucrase and isomaltase are enzymic activities of a single protein that is cleaved into two functional subunits, which remain associated in the cell membrane and form the sucrase-isomaltase (SI) complex. In contrast, maltase is one of two enzymic activities of the single membrane protein maltase-glucoamylase (MGA) that does not get cleaved. Its second enzymic activity, glucoamylase, cleaves α(1→4) glycosidic bonds in dextrins. D. Intestinal absorption of monosaccharides


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Biochemistry_Lippinco. D. Intestinal absorption of monosaccharides The upper jejunum absorbs the bulk of the monosaccharide products of digestion. However, different sugars have different mechanisms of absorption (Fig. 7.10). For example, galactose and glucose are taken into enterocytes by secondary active transport that requires a concurrent uptake (symport) of sodium (Na+) ions. The transport protein is the sodium-dependent glucose cotransporter 1 (SGLT-1). [Note: Sugar transport is driven by the Na+ gradient created by the Na+-potassium (K+) ATPase that moves Na+ out of the enterocyte and K+ in (see Fig. 7.10).] Fructose absorption utilizes an energy-and Na+-independent monosaccharide transporter (GLUT-5). All three monosaccharides are transported from the enterocytes into the portal circulation by yet another transporter, GLUT-2. [Note: See p. 97 for a discussion of these transporters.] E. Abnormal degradation of disaccharides


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Biochemistry_Lippinco. E. Abnormal degradation of disaccharides The overall process of carbohydrate digestion and absorption is so efficient in healthy individuals that ordinarily all digestible dietary carbohydrate is absorbed by the time the ingested material reaches the lower jejunum. However, because only monosaccharides are absorbed, any deficiency (genetic or acquired) in a specific disaccharidase activity of the intestinal mucosa causes the passage of undigested carbohydrate into the large intestine. As a consequence of the presence of this osmotically active material, water is drawn from the mucosa into the large intestine, causing osmotic diarrhea. This is reinforced by the bacterial fermentation of the remaining carbohydrate to two-and three-carbon compounds (which are also osmotically active) plus large volumes of carbon dioxide and hydrogen gas (H2), causing abdominal cramps, diarrhea, and flatulence. 1.


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Biochemistry_Lippinco. 1. Digestive enzyme deficiencies: Genetic deficiencies of the individual disaccharidases result in disaccharide intolerance. Alterations in disaccharide degradation can also be caused by a variety of intestinal diseases, malnutrition, and drugs that injure the mucosa of the small intestine. For example, brush border enzymes are rapidly lost in normal individuals with severe diarrhea, causing a temporary, acquired enzyme deficiency. Therefore, patients suffering or recovering from such a disorder cannot drink or eat significant amounts of dairy products or sucrose without exacerbating the diarrhea. 2.


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Biochemistry_Lippinco. Lactose intolerance: Over 60% of the world’s adults are lactose intolerant (Fig. 7.11). This is particularly manifested in certain populations. For example, up to 90% of adults of African or Asian descent are lactase deficient. Consequently, they are less able to metabolize lactose than are individuals of Northern European origin. The age-dependent loss of lactase activity starting at approximately age 2 years represents a reduction in the amount of enzyme produced. It is thought to be caused by small variations in the DNA sequence of a region on chromosome 2 that controls expression of the gene for lactase, also on chromosome 2. Treatment for this disorder is to reduce consumption of milk; eat yogurts and some cheeses (bacterial action and aging process decrease lactose content) as well as green vegetables, such as broccoli, to ensure adequate calcium intake; use lactase-treated products; or take lactase in pill form prior to eating. [Note: Because the loss of lactase is the norm for


Biochemistry_Lippinco

Biochemistry_Lippinco. green vegetables, such as broccoli, to ensure adequate calcium intake; use lactase-treated products; or take lactase in pill form prior to eating. [Note: Because the loss of lactase is the norm for most of the world’s adults, use of the terms adult-type hypolactasia or lactase nonpersistence rather than lactose intolerance is becoming more common.] Rare cases of congenital lactase deficiency are known.


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Biochemistry_Lippinco. 3. Congenital sucrase-isomaltase deficiency: This autosomal-recessive disorder results in an intolerance of ingested sucrose. Congenital SI deficiency has a prevalence of 1:5,000 in individuals of European descent and appears to be much more common (up to 1:20) in the Inuit people of Greenland and Canada. Treatment includes the dietary restriction of sucrose and enzyme replacement therapy. 4. Diagnosis: Identification of a specific enzyme deficiency can be obtained by performing oral tolerance tests with the individual disaccharides. Measurement of H2 in the breath is a reliable test for determining the amount of ingested carbohydrate not absorbed by the body, but which is metabolized instead by the intestinal flora (see Fig. 7.11). IV. CHAPTER SUMMARY


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Biochemistry_Lippinco. IV. CHAPTER SUMMARY Monosaccharides (Fig. 7.12) containing an aldehyde group are called aldoses, and those with a keto group are called ketoses. Disaccharides, oligosaccharides, and polysaccharides consist of monosaccharides linked by glycosidic bonds. Compounds with the same chemical formula but different structures are called isomers. Two monosaccharide isomers differing in configuration around one specific carbon atom (not the carbonyl carbon) are defined as epimers. In enantiomers (mirror images), the members of the sugar pair are designated as D-and L-isomers. When the aldehyde group on an acyclic sugar gets oxidized as a chromogenic agent gets reduced, that sugar is a reducing sugar. When a sugar cyclizes, an anomeric carbon is created from the carbonyl carbon of the aldehyde or keto group. The sugar can have two configurations, forming α or β anomers. A sugar can have its anomeric carbon linked to an –NH2 or an –


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Biochemistry_Lippinco. OH group on another structure through N-and O-glycosidic bonds, respectively. Salivary α-amylase initiates digestion of dietary polysaccharides (for example, starch or glycogen), producing oligosaccharides. Pancreatic α-amylase continues the process. The final digestive processes occur at the mucosal lining of the small intestine. Several disaccharidases (for example, lactase [β-galactosidase], sucrase, isomaltase, and maltase) produce monosaccharides (glucose, galactose, and fructose). These enzymes are transmembrane proteins of the luminal brush border of intestinal mucosal cells (enterocytes). Absorption of the monosaccharides requires specific transporters. If carbohydrate degradation is deficient (as a result of heredity, disease, or drugs that injure the intestinal mucosa), undigested carbohydrate will pass into the large intestine, where it can cause osmotic diarrhea. Bacterial fermentation of the material produces large volumes of carbon dioxide and hydrogen gas, causing


Biochemistry_Lippinco

Biochemistry_Lippinco. carbohydrate will pass into the large intestine, where it can cause osmotic diarrhea. Bacterial fermentation of the material produces large volumes of carbon dioxide and hydrogen gas, causing abdominal cramps, diarrhea, and flatulence. Lactose intolerance, primarily caused by the age-dependent loss of lactase (adult-type hypolactasia), is by far the most common of these deficiencies.


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Biochemistry_Lippinco. Choose the ONE best answer. .1. Which of the following statements best describes glucose? A. It is a C-4 epimer of galactose. B. It is a ketose and usually exists as a furanose ring in solution. C. It is produced from dietary starch by the action of α-amylase. D. It is utilized in biological systems only in the L-isomeric form. Correct answer = A. Because glucose and galactose differ only in configuration around carbon 4, they are C-4 epimers that are interconvertible by the action of an epimerase. Glucose is an aldose sugar that typically exists as a pyranose ring in solution. Fructose, however, is a ketose with a furanose ring. α-Amylase does not produce monosaccharides. The D-isomeric form of carbohydrates is the form typically found in biologic systems, in contrast to amino acids that typically are found in the L-isomeric form.


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Biochemistry_Lippinco. .2. A young man entered his physician’s office complaining of bloating and diarrhea. His eyes were sunken, and the physician noted additional signs of dehydration. The patient’s temperature was normal. He explained that the episode had occurred following a birthday party at which he had participated in an ice cream–eating contest. The patient reported prior episodes of a similar nature following ingestion of a significant amount of dairy products. This clinical picture is most probably due to a deficiency in the activity of: A. isomaltase. B. lactase. C. pancreatic α-amylase. D. salivary α-amylase. E. sucrase. Correct answer = B. The physical symptoms suggest a deficiency in an enzyme responsible for carbohydrate degradation. The symptoms observed following the ingestion of dairy products suggest that the patient is deficient in lactase as a result of the age-dependent reduction in expression of the enzyme.


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Biochemistry_Lippinco. .3. Routine examination of the urine of an asymptomatic pediatric patient showed a positive reaction with Clinitest (a copper reduction method of detecting reducing sugars) but a negative reaction with the glucose oxidase test for detecting glucose. Using these data, show on the chart below which of the sugars could (YES) or could not (NO) be present in the urine of this individual. Each of the listed sugars, except for sucrose and glucose, could be present in the urine of this individual. Clinitest is a nonspecific test that produces a change in color if urine is positive for reducing substances such as reducing sugars (fructose, galactose, glucose, lactose, xylulose). Because sucrose is not a reducing sugar, it is not detected by Clinitest. The glucose oxidase test will detect only glucose, and it cannot detect other sugars. The negative glucose oxidase test coupled with a positive reducing sugar test means that glucose cannot be the reducing sugar in the patient’s urine.


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Biochemistry_Lippinco. .4. Why are α-glucosidase inhibitors that are taken with meals, such as acarbose and miglitol, used in the treatment of diabetes? What effect should these drugs have on the digestion of lactose? α-Glucosidase inhibitors slow the production of glucose from dietary carbohydrates, thereby reducing the postprandial rise in blood glucose and facilitating better blood glucose control in diabetic patients. These drugs have no effect on lactose digestion because the disaccharide lactose contains a βglycosidic bond, not an α-glycosidic bond. Introduction to Metabolism and Glycolysis 8 For additional ancillary materials related to this chapter, please visit thePoint. I. METABOLISM OVERVIEW


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Biochemistry_Lippinco. In Chapter 5, individual enzymic reactions were analyzed in an effort to explain the mechanisms of catalysis. However, in cells, these reactions rarely occur in isolation. Instead, they are organized into multistep sequences called pathways, such as that of glycolysis (Fig. 8.1). In a pathway, the product of one reaction serves as the substrate of the subsequent reaction. Most pathways can be classified as either catabolic (degradative) or anabolic (synthetic). Catabolic pathways break down complex molecules, such as proteins, polysaccharides, and lipids, to a few simple molecules (for example, carbon dioxide, ammonia, and water). Anabolic pathways form complex end products from simple precursors, for example, the synthesis of the polysaccharide glycogen from glucose. [Note: Pathways that regenerate a component are called cycles.] Different pathways can intersect, forming an integrated and purposeful network of chemical reactions. Metabolism is the sum of all the chemical changes


Biochemistry_Lippinco

Biochemistry_Lippinco. that regenerate a component are called cycles.] Different pathways can intersect, forming an integrated and purposeful network of chemical reactions. Metabolism is the sum of all the chemical changes occurring in a cell, a tissue, or the body. The next several chapters focus on the central metabolic pathways that are involved in synthesizing and degrading carbohydrates, lipids, and amino acids.


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Biochemistry_Lippinco. A. Metabolic map Metabolism is best understood by examining its component pathways. Each pathway is composed of multienzyme sequences, and each enzyme, in turn, may exhibit important catalytic or regulatory features. A metabolic map containing the important central pathways of energy metabolism is presented in Figure 8.2. This “big picture” view of metabolism is useful in tracing connections between pathways, visualizing the purposeful movement of metabolic intermediates (metabolites), and depicting the effect on the flow of intermediates if a pathway is blocked (for example, by a drug or an inherited deficiency of an enzyme). [Note: The metabolome is the full complement of metabolites in an organism.] Throughout the next three units of this book, each pathway under discussion will be repeatedly featured as part of the major metabolic map shown in Figure 8.2. intermediates of protein metabolism. UDP = uridine diphosphate; P = phosphate; -


Biochemistry_Lippinco

Biochemistry_Lippinco. intermediates of protein metabolism. UDP = uridine diphosphate; P = phosphate; - CoA = coenzyme A; CO2 = carbon dioxide; HCO3 = bicarbonate; NH3 = ammonia. B. Catabolic pathways Catabolic reactions serve to capture chemical energy in the form of ATP from the degradation of energy-rich fuel molecules. ATP generation by degradation of complex molecules occurs in three stages, as shown in Figure 8.3. [Note: Catabolic pathways are typically oxidative and require oxidized coenzymes such as nicotinamide adenine dinucleotide (NAD+).] Catabolism also allows molecules in the diet (or nutrient molecules stored in cells) to be converted into basic building blocks needed for the synthesis of complex molecules. Catabolism, then, is a convergent process (that is, a wide variety of molecules are transformed into a few common end products). 1.


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Biochemistry_Lippinco. 1. Hydrolysis of complex molecules: In the first stage, complex molecules are broken down into their component building blocks. For example, proteins are degraded to amino acids, polysaccharides to monosaccharides, and fats (triacylglycerols) to free fatty acids and glycerol. 2. Conversion of building blocks to simple intermediates: In the second stage, these diverse building blocks are further degraded to acetyl coenzyme A (CoA) and a few other simple molecules. Some energy is captured as ATP, but the amount is small compared with the energy produced during the third stage of catabolism. 3. Oxidation of acetyl coenzyme A: The tricarboxylic acid (TCA) cycle (see p. 109) is the final common pathway in the oxidation of fuel molecules that produce acetyl CoA. Oxidation of acetyl CoA generates large amounts of ATP via oxidative phosphorylation as electrons flow from NADH and flavin adenine dinucleotide (FADH2) to oxygen ([O2] see p. 73). C. Anabolic pathways


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Biochemistry_Lippinco. 73). C. Anabolic pathways In contrast to catabolism, anabolism is a divergent process in which a few biosynthetic precursors (such as amino acids) form a wide variety of polymeric, or complex, products (such as proteins [Fig. 8.4]). Anabolic reactions require energy (are endergonic), which is generally provided by the hydrolysis of ATP to adenosine diphosphate (ADP) and inorganic phosphate (Pi). [Note: Catabolic reactions generate energy (are exergonic).] Anabolic reactions often involve chemical reductions in which the reducing power is most frequently provided by the electron donor NADPH (phosphorylated NADH, see p. 147). II. METABOLISM REGULATION


Biochemistry_Lippinco

Biochemistry_Lippinco. Anabolic reactions often involve chemical reductions in which the reducing power is most frequently provided by the electron donor NADPH (phosphorylated NADH, see p. 147). II. METABOLISM REGULATION The pathways of metabolism must be coordinated so that the production of energy or the synthesis of end products meets the needs of the cell. Furthermore, individual cells function as part of a community of interacting tissues, not in isolation. Thus, a sophisticated communication system has evolved to coordinate the functions of the body. Regulatory signals that inform an individual cell of the metabolic state of the body as a whole include hormones, neurotransmitters, and the availability of nutrients. These, in turn, influence signals generated within the cell (Fig. 8.5). A. Intracellular communication


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Biochemistry_Lippinco. A. Intracellular communication The rate of a metabolic pathway can respond to regulatory signals that arise from within the cell. For example, the rate may be influenced by the availability of substrates, product inhibition, or alterations in the levels of allosteric activators or inhibitors. These intracellular signals typically elicit rapid responses and are important for the moment-to-moment regulation of metabolism. B. Intercellular communication


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Biochemistry_Lippinco. B. Intercellular communication The ability to respond to intercellular signals is essential for the development and survival of organisms. Signaling between cells provides for long-range integration of metabolism and usually results in a response, such as a change in gene expression, that is slower than is seen with intracellular signals. Communication between cells can be mediated, for example, by surface-to-surface contact and, in some tissues, by formation of gap junctions, allowing direct communication between the cytoplasms of adjacent cells. However, for energy metabolism, the most important route of communication is chemical signaling between cells by blood-borne hormones or by neurotransmitters. C. Second messenger systems


Biochemistry_Lippinco

Biochemistry_Lippinco. Hormones and neurotransmitters can be thought of as signals and their receptors as signal detectors. Receptors respond to a bound ligand by initiating a series of reactions that ultimately result in specific intracellular responses. Second messenger molecules, so named because they intervene between the original extracellular messenger (the neurotransmitter or hormone) and the ultimate intracellular effect, are part of the cascade of events that converts (transduces) ligand binding into a response. Two of the most widely recognized second messenger systems are the calcium/phosphatidylinositol system (see p. 205) and the adenylyl cyclase (adenylate cyclase) system, which is particularly important in regulating the pathways of intermediary metabolism. Both involve the binding of ligands, such as epinephrine or glucagon, to specific G protein–coupled receptors (GPCR) on the cell (plasma) membrane. GPCR are characterized by an extracellular ligand-binding domain, seven transmembrane α


Biochemistry_Lippinco

Biochemistry_Lippinco. such as epinephrine or glucagon, to specific G protein–coupled receptors (GPCR) on the cell (plasma) membrane. GPCR are characterized by an extracellular ligand-binding domain, seven transmembrane α helices, and an intracellular domain that interacts with trimeric G proteins (Fig. 8.6). [Note: Insulin, another key regulator of metabolism, binds a membrane tyrosine kinase receptor (see p. 311) and not a GPCR.]


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Biochemistry_Lippinco. D. Adenylyl cyclase The recognition of a chemical signal by some GPCR, such as the β-and α2adrenergic receptors, triggers either an increase or a decrease in the activity of adenylyl cyclase (AC). This is a membrane-bound enzyme that converts ATP to 3ʹ,5ʹ-adenosine monophosphate (cyclic AMP, or cAMP). The chemical signals are most often hormones or neurotransmitters, each of which binds to a unique type of GPCR. Therefore, tissues that respond to more than one signal must have several different GPCR, each of which can be linked to AC.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. Guanosine triphosphate–dependent regulatory proteins: The effect of the activated, occupied GPCR on second messenger formation is indirect, mediated by specialized trimeric proteins (α, β, and γ subunits) of the cell membrane. These proteins, referred to as G proteins because the α subunit binds guanosine di-or triphosphates (GDP or GTP), form a link in the chain of communication between the receptor and AC. In the inactive form of a G protein, the α subunit is bound to GDP (Fig. 8.7). Ligand binding causes a conformational change in the receptor, triggering replacement of this GDP with GTP. The GTP-bound form of the α subunit dissociates from the βγ subunits and moves to AC, affecting enzyme activity. Many molecules of active Gα protein are formed by one activated receptor. [Note: The ability of a hormone or neurotransmitter to stimulate or inhibit AC depends on the type of Gα protein that is linked to the receptor. One type, designated Gs, stimulates AC (see Fig. 8.7), whereas


Biochemistry_Lippinco

Biochemistry_Lippinco. ability of a hormone or neurotransmitter to stimulate or inhibit AC depends on the type of Gα protein that is linked to the receptor. One type, designated Gs, stimulates AC (see Fig. 8.7), whereas another type, designated Gi, inhibits the enzyme (not shown).] The actions of the Gα–GTP complex are short-lived because Gα has an inherent GTPase activity, resulting in the rapid hydrolysis of GTP to GDP. This causes inactivation of Gα, its dissociation from AC, and its reassociation with the βγ dimer.


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Biochemistry_Lippinco. Toxins from Vibrio cholerae (cholera) and Bordetella pertussis (whooping cough) cause inappropriate activation of AC through covalent modification (ADP-ribosylation) of different G proteins. With cholera, the GTPase activity of Gαs is inhibited in intestinal cells. With whooping cough, Gαi is inactivated in respiratory tract cells.


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Biochemistry_Lippinco. 2. Protein kinases: The next step in the cAMP second messenger system is the activation of a family of enzymes called cAMP-dependent protein kinases such as protein kinase A (PKA), as shown in Figure 8.8. cAMP activates PKA by binding to its two regulatory subunits, causing the release of its two catalytically active subunits. These subunits transfer phosphate from ATP to specific serine or threonine residues of protein substrates. The phosphorylated proteins may act directly on the cell’s ion channels or, if enzymes, may become activated or inhibited. [Note: Several types of protein kinases are not cAMP dependent, for example, protein kinase C, described on p. 205.] 3. Protein phosphatases: The phosphate groups added to proteins by protein kinases are removed by protein phosphatases, enzymes that hydrolytically cleave phosphate esters (see Fig. 8.8). This insures that changes in protein activity induced by phosphorylation are not permanent. 4.


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Biochemistry_Lippinco. 4. cAMP hydrolysis: cAMP is rapidly hydrolyzed to 5ʹ-AMP by cAMP phosphodiesterase that cleaves the cyclic 3ʹ,5ʹ-phosphodiester bond. 5ʹAMP is not an intracellular signaling molecule. Therefore, the effects of neurotransmitter-or hormone-mediated increases of cAMP are rapidly terminated if the extracellular signal is removed. [Note: cAMP phosphodiesterase is inhibited by caffeine, a methylxanthine derivative.] III. GLYCOLYSIS OVERVIEW


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Biochemistry_Lippinco. The glycolytic pathway is used by all tissues for the oxidation of glucose to provide energy (as ATP) and intermediates for other metabolic pathways. Glycolysis is at the hub of carbohydrate metabolism because virtually all sugars, whether arising from the diet or from catabolic reactions in the body, can ultimately be converted to glucose (Fig. 8.9A). Pyruvate is the end product of glycolysis in cells with mitochondria and an adequate supply of O2. This series of ten reactions is called aerobic glycolysis because O2 is required to reoxidize the NADH formed during the oxidation of glyceraldehyde 3-phosphate (Fig. 8.9B). Aerobic glycolysis sets the stage for the oxidative decarboxylation of pyruvate to acetyl CoA, a major fuel of the TCA cycle. Alternatively, pyruvate is reduced to lactate as NADH is oxidized to NAD+ (Fig. 8.9C). This conversion of glucose to lactate is called anaerobic glycolysis because it can occur without the participation of O2. Anaerobic glycolysis allows the


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Biochemistry_Lippinco. as NADH is oxidized to NAD+ (Fig. 8.9C). This conversion of glucose to lactate is called anaerobic glycolysis because it can occur without the participation of O2. Anaerobic glycolysis allows the production of ATP in tissues that lack mitochondria (for example, red blood cells [RBC] and parts of the eye) or in cells deprived of sufficient O2 (hypoxia).


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Biochemistry_Lippinco. IV. GLUCOSE TRANSPORT INTO CELLS Glucose cannot diffuse directly into cells but enters by one of two transport systems: a sodium (Na+)-and ATP-independent transport system or a Na+-and ATP-dependent cotransport system. A. Sodium-and ATP-independent transport system This passive system is mediated by a family of 14 glucose transporter (GLUT) isoforms found in cell membranes. They are designated GLUT-1 to GLUT-14. These monomeric protein transporters exist in the membrane in two conformational states (Fig. 8.10). Extracellular glucose binds to the transporter, which then alters its conformation, transporting glucose across the cell membrane via facilitated diffusion. Because GLUT transport one molecule at a time, they are uniporters. 1. Tissue specificity: GLUT display a tissue-specific pattern of expression.


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Biochemistry_Lippinco. 1. Tissue specificity: GLUT display a tissue-specific pattern of expression. For example, GLUT-3 is the primary isoform in neurons. GLUT-1 is abundant in RBC and the blood–brain barrier but is low in adult muscle, whereas GLUT-4 is abundant in muscle and adipose tissue. [Note: The number of GLUT-4 transporters active in these tissues is increased by insulin. (See p. 311 for a discussion of insulin and glucose transport.)] GLUT-2 is abundant in the liver, kidneys, and pancreatic β cells. The other GLUT isoforms also have tissue-specific distributions. 2.


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Biochemistry_Lippinco. 2. Specialized functions: In facilitated diffusion, transporter-mediated glucose movement is down a concentration gradient (that is, from a high concentration to a lower one, therefore requiring no energy). For example, GLUT-1, GLUT-3, and GLUT-4 are primarily involved in glucose uptake from the blood. In contrast, GLUT-2, in the liver and kidneys, can either transport glucose into these cells when blood glucose levels are high or transport glucose from these cells when blood glucose levels are low (for example, during fasting). GLUT-5 is unusual in that it is the primary transporter for fructose (not glucose) in the small intestine and the testes (see p. 87). B. Sodium-and ATP-dependent cotransport system


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Biochemistry_Lippinco. B. Sodium-and ATP-dependent cotransport system This energy-requiring process transports glucose against (up) its concentration gradient (that is, from low extracellular concentrations to higher intracellular concentrations) as Na+ is transported down its electrochemical gradient. [Note: The gradient is created by the Na+potassium (K+) ATPase (see Fig. 7.10, p. 87).] Because this secondary active transport process requires the concurrent uptake (symport) of Na+, the transporter is a sodium-dependent glucose cotransporter (SGLT). This type of cotransport occurs in the epithelial cells of the intestine (see p. 87), renal tubules, and choroid plexus. [Note: The choroid plexus, part of the blood–brain barrier, also contains GLUT-1.] V. GLYCOLYSIS REACTIONS


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Biochemistry_Lippinco. V. GLYCOLYSIS REACTIONS The conversion of glucose to pyruvate occurs in two stages (Fig. 8.11). The first five reactions of glycolysis correspond to an energy-investment phase in which the phosphorylated forms of intermediates are synthesized at the expense of ATP. The subsequent reactions of glycolysis constitute an energy-generation phase in which a net of two molecules of ATP are formed by substrate-level phosphorylation (see p. 102) per glucose molecule metabolized. A. Glucose phosphorylation Phosphorylated sugar molecules do not readily penetrate cell membranes because there are no specific transmembrane carriers for these compounds and because they are too polar to diffuse through the lipid core of membranes. Therefore, the irreversible phosphorylation of glucose (Fig.


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Biochemistry_Lippinco. 8.12) effectively traps the sugar as cytosolic glucose 6-phosphate and commits it to further metabolism in the cell. Mammals have four isozymes (I–IV) of the enzyme hexokinase that catalyze the phosphorylation of glucose to glucose 6-phosphate.


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Biochemistry_Lippinco. 1. Hexokinases I–III: In most tissues, glucose phosphorylation is catalyzed by one of these isozymes of hexokinase, which is one of three regulatory enzymes of glycolysis (along with phosphofructokinase and pyruvate kinase). They are inhibited by the reaction product glucose 6-phosphate, which accumulates when further metabolism of this hexose phosphate is reduced. Hexokinases I–III have a low Michaelis constant (Km) and, therefore, a high affinity (see p. 59) for glucose. This permits the efficient phosphorylation and subsequent metabolism of glucose even when tissue concentrations of glucose are low (Fig. 8.13). However, because these isozymes have a low maximal velocity ([Vmax] see p. 57) for glucose, they do not sequester (trap) cellular phosphate in the form of phosphorylated glucose or phosphorylate more glucose than the cell can use. [Note: These isozymes have broad substrate specificity and are able to phosphorylate several hexoses in addition to glucose.] 2. Hexokinase IV: In


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Biochemistry_Lippinco. or phosphorylate more glucose than the cell can use. [Note: These isozymes have broad substrate specificity and are able to phosphorylate several hexoses in addition to glucose.] 2. Hexokinase IV: In liver parenchymal cells and pancreatic β cells, glucokinase (the hexokinase IV isozyme) is the predominant enzyme responsible for glucose phosphorylation. In β cells, glucokinase functions as a glucose sensor, determining the threshold for insulin secretion (see p. 309). [Note: Hexokinase IV also serves as a glucose sensor in hypothalamic neurons, playing a key role in the adrenergic response to hypoglycemia (see p. 315).] In the liver, the enzyme facilitates glucose phosphorylation during hyperglycemia. Despite the popular but misleading name glucokinase, the sugar specificity of the enzyme is similar to that of other hexokinase isozymes.


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Biochemistry_Lippinco. a. Kinetics: Glucokinase differs from hexokinases I–III in several important properties. For example, it has a much higher Km, requiring a higher glucose concentration for half-saturation (see Fig. 8.13). Thus, glucokinase functions only when the intracellular concentration of glucose in the hepatocyte is elevated such as during the brief period following consumption of a carbohydrate-rich meal, when high levels of glucose are delivered to the liver via the portal vein. Glucokinase has a high Vmax, allowing the liver to effectively remove the flood of glucose delivered by the portal blood. This prevents large amounts of glucose from entering the systemic circulation following such a meal, thereby minimizing hyperglycemia during the absorptive period. [Note: GLUT-2 insures that blood glucose equilibrates rapidly across the hepatocyte membrane.] b. Regulation: Glucokinase activity is not directly inhibited by glucose 6phosphate as are the other hexokinases. Instead, it is indirectly


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Biochemistry_Lippinco. equilibrates rapidly across the hepatocyte membrane.] b. Regulation: Glucokinase activity is not directly inhibited by glucose 6phosphate as are the other hexokinases. Instead, it is indirectly inhibited by fructose 6-phosphate (which is in equilibrium with glucose 6-phosphate, a product of glucokinase) and is indirectly stimulated by glucose (a substrate of glucokinase). Regulation is achieved by reversible binding to the hepatic protein glucokinase regulatory protein (GKRP). In the presence of fructose 6-phosphate, glucokinase binds tightly to GKRP and is translocated to the nucleus, thereby rendering the enzyme inactive (Fig. 8.14). When glucose levels in the blood (and also in the hepatocyte, as a result of GLUT-2) increase, glucokinase is released from GKRP, and the enzyme reenters the cytosol where it phosphorylates glucose to glucose 6-phosphate. [Note: GKRP is a competitive inhibitor of glucose use by glucokinase.]


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Biochemistry_Lippinco. Glucokinase functions as a glucose sensor in blood glucose homeostasis. Inactivating mutations of glucokinase are the cause of a rare form of diabetes, maturity onset diabetes of the young type 2 (MODY 2) that is characterized by impaired insulin secretion and hyperglycemia. B. Glucose 6-phosphate isomerization The isomerization of glucose 6-phosphate to fructose 6-phosphate is catalyzed by phosphoglucose isomerase (Fig. 8.15). The reaction is readily reversible and is not a rate-limiting or regulated step. C. Fructose 6-phosphate phosphorylation The irreversible phosphorylation reaction catalyzed by phosphofructokinase-1 (PFK-1) is the most important control point and the rate-limiting and committed step of glycolysis (Fig. 8.16). PFK-1 is controlled by the available concentrations of the substrates ATP and fructose 6-phosphate as well as by other regulatory molecules. 1.


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Biochemistry_Lippinco. 1. Regulation by intracellular energy levels: PFK-1 is inhibited allosterically by elevated levels of ATP, which act as an energy-rich signal indicating an abundance of high-energy compounds. Elevated levels of citrate, an intermediate in the TCA cycle (see p. 111), also inhibit PFK-1. [Note: Inhibition by citrate favors the use of glucose for glycogen synthesis (see p. 126).] Conversely, PFK-1 is activated allosterically by high concentrations of AMP, which signal that the cell’s energy stores are depleted. 2.


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Biochemistry_Lippinco. Regulation by fructose 2,6-bisphosphate: Fructose 2,6-bisphosphate is the most potent activator of PFK-1 (see Fig. 8.16) and is able to activate the enzyme even when ATP levels are high. It is formed from fructose 6phosphate by phosphofructokinase-2 (PFK-2). Unlike PFK-1, PFK-2 is a bifunctional protein that has both the kinase activity that produces fructose 2,6-bisphosphate and the phosphatase activity that dephosphorylates fructose 2,6-bisphosphate to fructose 6-phosphate. In the liver isozyme, phosphorylation of PFK-2 inactivates the kinase domain and activates the phosphatase domain (Fig. 8.17). The opposite is seen in the cardiac isozyme. Skeletal PFK-2 is not covalently regulated. [Note: Fructose 2,6-bisphosphate is an inhibitor of fructose 1,6-bisphosphatase, an enzyme of gluconeogenesis (see p. 121). The reciprocal actions of fructose 2,6-bisphosphate on glycolysis (activation) and gluconeogenesis (inhibition) insure that both pathways are not fully active at the same time,


Biochemistry_Lippinco

Biochemistry_Lippinco. (see p. 121). The reciprocal actions of fructose 2,6-bisphosphate on glycolysis (activation) and gluconeogenesis (inhibition) insure that both pathways are not fully active at the same time, preventing a futile cycle of glucose oxidation to pyruvate followed by glucose resynthesis from pyruvate.] a.


Biochemistry_Lippinco

Biochemistry_Lippinco. During the well-fed state: Decreased levels of glucagon and elevated levels of insulin (such as occur following a carbohydrate-rich meal) cause an increase in hepatic fructose 2,6-bisphos-phate (PFK-2 is dephosphorylated) and, thus, in the rate of glycolysis (see Fig. 8.17). Therefore, fructose 2,6-bisphosphate acts as an intracellular signal of glucose abundance. b. During fasting: By contrast, the elevated levels of glucagon and low levels of insulin that occur during fasting (see p. 327) cause a decrease in hepatic fructose 2,6-bisphosphate (PFK-2 is phosphorylated). This results in inhibition of glycolysis and activation of gluconeogenesis. D. Fructose 1,6-bisphosphate cleavage


Biochemistry_Lippinco

Biochemistry_Lippinco. D. Fructose 1,6-bisphosphate cleavage Aldolase cleaves fructose 1,6-bisphosphate to dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (see Fig. 8.16). The reaction is reversible and not regulated. [Note: Aldolase B, the hepatic isoform, also cleaves fructose 1-phosphate and functions in dietary fructose metabolism (see p. 138).] E. Dihydroxyacetone phosphate isomerization Triose phosphate isomerase interconverts DHAP and glyceraldehyde 3phosphate (see Fig. 8.16). DHAP must be isomerized to glyceraldehyde 3phosphate for further metabolism by the glycolytic pathway. This isomerization results in the net production of two molecules of glyceraldehyde 3-phosphate from the cleavage products of fructose 1,6bisphosphate. [Note: DHAP is utilized in triacylglycerol synthesis (see p. 188).] F. Glyceraldehyde 3-phosphate oxidation


Biochemistry_Lippinco

Biochemistry_Lippinco. F. Glyceraldehyde 3-phosphate oxidation The conversion of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate (1,3-BPG) by glyceraldehyde 3-phosphate dehydrogenase is the first oxidation-reduction reaction of glycolysis (Fig. 8.18). [Note: Because there is a limited amount of NAD+ in the cell, the NADH formed by the dehydrogenase reaction must be oxidized for glycolysis to continue. Two major mechanisms for oxidizing NADH to NAD+ are the reduction of pyruvate to lactate by lactate dehydrogenase (LDH) (anaerobic, see p. 96) and the electron transport chain ([ETC] aerobic, see p. 74). Because NADH cannot cross the inner mitochondrial membrane, the ETC requires the malate-aspartate and glycerol 3-phosphate substrate shuttles to move NADH reducing equivalents into the mitochondrial matrix (see p. 79).] 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1,3-Bisphosphoglycerate synthesis: The oxidation of the aldehyde group of glyceraldehyde 3-phosphate to a carboxyl group is coupled to the attachment of Pi to the carboxyl group. This phosphate group, linked to carbon 1 of the 1,3-BPG product by a high-energy bond (see p. 73), conserves much of the free energy (see p. 69) produced by the oxidation of glyceraldehyde 3-phosphate. This high-energy phosphate drives ATP synthesis in the next reaction of glycolysis. 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. Arsenic poisoning: The toxicity of arsenic is due primarily to the inhibition by trivalent arsenic (arsenite) of enzymes such as the pyruvate dehydrogenase complex (PDHC), which require lipoic acid as a coenzyme (see p. 110). However, pentavalent arsenic (arsenate) can prevent net ATP and NADH production by glycolysis without inhibiting the pathway itself. It does so by competing with Pi as a substrate for glyceraldehyde 3-phosphate dehydrogenase, forming a complex that spontaneously hydrolyzes to form 3-phosphoglycerate (see Fig. 8.18). By bypassing the synthesis of and phosphate transfer from 1,3-BPG, the cell is deprived of energy usually obtained from the glycolytic pathway. [Note: Arsenate also competes with Pi binding to the F1 domain of ATP synthase (see p. 78), resulting in formation of ADP-arsenate that is rapidly hydrolyzed.] 3. 2,3-Bisphosphoglycerate synthesis in RBC: Some of the 1,3-BPG is converted to 2,3-BPG by the action of bisphosphoglycerate mutase (see Fig. 8.18).


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Biochemistry_Lippinco. of ADP-arsenate that is rapidly hydrolyzed.] 3. 2,3-Bisphosphoglycerate synthesis in RBC: Some of the 1,3-BPG is converted to 2,3-BPG by the action of bisphosphoglycerate mutase (see Fig. 8.18). 2,3-BPG, which is found in only trace amounts in most cells, is present at high concentration in RBC and serves to increase O2 delivery (see p. 31). 2,3-BPG is hydrolyzed by a phosphatase to 3phosphoglycerate, which is also an intermediate in glycolysis (see Fig. 8.18). In the RBC, glycolysis is modified by inclusion of these shunt reactions.


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Biochemistry_Lippinco. G. 3-Phosphoglycerate synthesis and ATP production When 1,3-BPG is converted to 3-phosphoglycerate, the high-energy phosphate group of 1,3-BPG is used to synthesize ATP from ADP (see Fig. 8.18). This reaction is catalyzed by phosphoglycerate kinase, which, unlike most other kinases, is physiologically reversible. Because two molecules of 1,3-BPG are formed from each glucose molecule, this kinase reaction replaces the two ATP molecules consumed by the earlier formation of glucose 6-phosphate and fructose 1,6-bisphosphate. [Note: This reaction is an example of substrate-level phosphorylation, in which the energy needed for the production of a high-energy phosphate comes from a substrate rather than from the ETC (see J. below and p. 113 for other examples).] H. Phosphate group shift The shift of the phosphate group from carbon 3 to carbon 2 of phosphoglycerate by phosphoglycerate mutase is freely reversible. I. 2-Phosphoglycerate dehydration


Biochemistry_Lippinco

Biochemistry_Lippinco. H. Phosphate group shift The shift of the phosphate group from carbon 3 to carbon 2 of phosphoglycerate by phosphoglycerate mutase is freely reversible. I. 2-Phosphoglycerate dehydration The dehydration of 2-phosphoglycerate by enolase redistributes the energy within the substrate, forming phosphoenolpyruvate (PEP), which contains a high-energy enol phosphate (see Fig. 8.18). The reaction is reversible, despite the high-energy nature of the product. [Note: Fluoride inhibits enolase, and water fluoridation reduces lactate production by mouth bacteria, decreasing dental caries (see p. 405).] J. Pyruvate synthesis and ATP production The conversion of PEP to pyruvate, catalyzed by pyruvate kinase (PK), is the third irreversible reaction of glycolysis. The high-energy enol phosphate in PEP is used to synthesize ATP from ADP and is another example of substrate-level phosphorylation (see Fig. 8.18). 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. Feedforward regulation: PK is activated by fructose 1,6-bisphosphate, the product of the PFK-1 reaction. This feedforward (instead of the more usual feedback) regulation has the effect of linking the two kinase activities: increased PFK-1 activity results in elevated levels of fructose 1,6-bisphosphate, which activates PK. [Note: PK is inhibited by ATP.] 2. Covalent regulation in the liver: Phosphorylation by cAMP-dependent PKA leads to inactivation of the hepatic isozyme of PK (Fig. 8.19). When blood glucose levels are low, elevated glucagon increases the intracellular level of cAMP, which causes the phosphorylation and inactivation of PK in the liver only. Therefore, PEP is unable to continue in glycolysis and, instead, enters the gluconeogenesis pathway. This partly explains the observed inhibition of hepatic glycolysis and stimulation of gluconeogenesis by glucagon. Dephosphorylation of PK by a phosphatase results in reactivation of the enzyme. diphosphate.


Biochemistry_Lippinco

Biochemistry_Lippinco. 3. Pyruvate kinase deficiency: Because mature RBC lack mitochondria, they are completely dependent on glycolysis for ATP production. ATP is required to meet the metabolic needs of RBC and to fuel the ion pumps necessary for the maintenance of the flexible, biconcave shape that allows them to squeeze through narrow capillaries. The anemia observed in glycolytic enzyme deficiencies is a consequence of the reduced rate of glycolysis, leading to decreased ATP production by substrate-level phosphorylation. The resulting alterations in the RBC membrane lead to changes in cell shape and, ultimately, to phagocytosis by cells of the mononuclear phagocyte system, particularly splenic macrophages. The premature death and lysis of RBC result in mild-to-severe nonspherocytic hemolytic anemia, with the severe form requiring regular transfusions. Among patients with rare genetic defects of glycolytic enzymes, the majority has a deficiency in PK. [Note: Liver PK is encoded by the same gene as the RBC


Biochemistry_Lippinco

Biochemistry_Lippinco. severe form requiring regular transfusions. Among patients with rare genetic defects of glycolytic enzymes, the majority has a deficiency in PK. [Note: Liver PK is encoded by the same gene as the RBC isozyme. However, liver cells show no effect because they can synthesize more PK and can also generate ATP by oxidative phosphorylation.] Severity depends both on the degree of enzyme deficiency (generally 5%–35% of normal levels) and on the extent to which RBC compensate by synthesizing increased levels of 2,3-BPG (see p. 31). Almost all individuals with PK deficiency have a mutant enzyme that shows altered kinetics or decreased stability (Fig. 8.20). Individuals heterozygous for PK deficiency have resistance to the most severe forms of malaria.


Biochemistry_Lippinco

Biochemistry_Lippinco. The tissue-specific expression of PK in RBC and the liver results from the use of different start sites in transcription (see p. 473) of the gene that encodes the enzyme. K. Pyruvate reduction to lactate Lactate, formed from pyruvate by LDH, is the final product of anaerobic glycolysis in eukaryotic cells (Fig. 8.21). Reduction to lactate is the major fate for pyruvate in tissues that are poorly vascularized (for example, the lens and cornea of the eye and the kidney medulla) or in RBC that lack mitochondria. 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. Lactate formation in muscle: In exercising skeletal muscle, NADH production (by glyceraldehyde 3-phosphate dehydrogenase and by the three NAD+-linked dehydrogenases of the TCA cycle, see p. 113) exceeds the oxidative capacity of the ETC. This results in an elevated NADH/NAD+ ratio, favoring reduction of pyruvate to lactate by LDH. Therefore, during intense exercise, lactate accumulates in muscle, causing a drop in the intracellular pH, potentially resulting in cramps. Much of this lactate eventually diffuses into the bloodstream and can be used by the liver to make glucose (see p. 118). 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Lactate utilization: The direction of the LDH reaction depends on the relative intracellular concentrations of pyruvate and lactate and on the ratio of NADH/NAD+. For example, in the liver and heart, this ratio is lower than in exercising muscle. Consequently, the liver and heart oxidize lactate (obtained from the blood) to pyruvate. In the liver, pyruvate is either converted to glucose by gluconeogenesis or converted to acetyl CoA that is oxidized in the TCA cycle. Heart muscle exclusively oxidizes lactate to carbon dioxide and water via the TCA cycle. 3.


Biochemistry_Lippinco

Biochemistry_Lippinco. Lactic acidosis: Elevated concentrations of lactate in the plasma, termed lactic acidosis (a type of metabolic acidosis), occur when there is a collapse of the circulatory system, such as with myocardial infarction, pulmonary embolism, and uncontrolled hemorrhage, or when an individual is in shock. The failure to bring adequate amounts of O2 to the tissues results in impaired oxidative phosphorylation and decreased ATP synthesis. To survive, the cells rely on anaerobic glycolysis for generating ATP, producing lactic acid as the end product. [Note: Production of even meager amounts of ATP may be lifesaving during the period required to reestablish adequate blood flow to the tissues.] The additional O2 required to recover from a period when O2 availability has been inadequate is termed the O2 debt. [Note: The O2 debt is often related to patient morbidity or mortality. In many clinical situations, measuring the blood levels of lactic acid allows the rapid, early detection of O2 debt in


Biochemistry_Lippinco

Biochemistry_Lippinco. O2 debt. [Note: The O2 debt is often related to patient morbidity or mortality. In many clinical situations, measuring the blood levels of lactic acid allows the rapid, early detection of O2 debt in patients and the monitoring of their recovery.]


Biochemistry_Lippinco

Biochemistry_Lippinco. L. Energy yield from glycolysis Despite the production of some ATP by substrate-level phosphorylation during glycolysis, the end product, pyruvate or lactate, still contains most of the energy originally contained in glucose. The TCA cycle is required to release that energy completely (see p. 109). 1. Anaerobic glycolysis: A net of two molecules of ATP are generated for each molecule of glucose converted to two molecules of lactate (Fig. 8.22). There is no net production or consumption of NADH. 2. Aerobic glycolysis: The generation of ATP is the same as in anaerobic glycolysis (that is, a net gain of two ATP per molecule of glucose). Two molecules of NADH are also produced per molecule of glucose. Ongoing aerobic glycolysis requires the oxidation of most of this NADH by the ETC, producing three ATP for each NADH molecule entering the chain (see p. 77). [Note: NADH cannot cross the inner mitochondrial membrane, and substrate shuttles are required (see p. 79).]


Biochemistry_Lippinco

VI. HORMONAL REGULATION

Biochemistry_Lippinco. VI. HORMONAL REGULATION


Biochemistry_Lippinco

Biochemistry_Lippinco. Regulation of the activity of the irreversible glycolytic enzymes by allosteric activation/inhibition or covalent phosphorylation/dephosphorylation is short term (that is, the effects occur over minutes or hours). Superimposed on these effects on the activity of preexisting enzyme molecules are the long-term hormonal effects on the number of new enzyme molecules. These hormonal effects can result in 10-to 20-fold increases in enzyme synthesis that typically occur over hours to days. Regular consumption of meals rich in carbohydrate or administration of insulin initiates an increase in the amount of glucokinase, PFK-1, and PK in the liver (Fig. 8.23). The change reflects an increase in gene transcription, resulting in increased enzyme synthesis. Increased availability of these three enzymes favors the conversion of glucose to pyruvate, a characteristic of the absorptive state (see p. 321). [Note: The transcriptional effects of insulin and carbohydrate (specifically glucose) are


Biochemistry_Lippinco

Biochemistry_Lippinco. enzymes favors the conversion of glucose to pyruvate, a characteristic of the absorptive state (see p. 321). [Note: The transcriptional effects of insulin and carbohydrate (specifically glucose) are mediated by the transcription factors sterol regulatory element–binding protein-1c and carbohydrate response element–binding protein, respectively. These factors also regulate transcription of genes involved in fatty acid synthesis (see p. 184).] Conversely, gene expression of the three enzymes is decreased when plasma glucagon is high and insulin is low (for example, as seen in fasting or diabetes).


Biochemistry_Lippinco

Biochemistry_Lippinco. VII. ALTERNATE FATES OF PYRUVATE Pyruvate can be metabolized to products other than lactate. A. Oxidative decarboxylation to acetyl CoA Oxidative decarboxylation of pyruvate by the PDHC is an important pathway in tissues with a high oxidative capacity such as cardiac muscle (Fig. 8.24). PDHC irreversibly converts pyruvate, the end product of aerobic glycolysis, into acetyl CoA, a TCA cycle substrate (see p. 109) and the carbon source for fatty acid synthesis (see p. 183). B. Carboxylation to oxaloacetate Carboxylation of pyruvate to oxaloacetate by pyruvate carboxylase is a biotin-dependent reaction (see Fig. 8.24). This irreversible reaction is important because it replenishes the TCA cycle intermediate and provides substrate for gluconeogenesis (see p. 118). C. Reduction to ethanol (microorganisms)


Biochemistry_Lippinco

Biochemistry_Lippinco. C. Reduction to ethanol (microorganisms) The reduction of pyruvate to ethanol occurs by the two reactions summarized in Figure 8.24. The decarboxylation of pyruvate to acetaldehyde by thiamine-requiring pyruvate decarboxylase occurs in yeast and certain other microorganisms but not in humans. VIII. CHAPTER SUMMARY


Biochemistry_Lippinco

Biochemistry_Lippinco. Most pathways can be classified as either catabolic (degrade complex molecules to a few simple products with ATP production) or anabolic (synthesize complex end products from simple precursors with ATP hydrolysis). The rate of a metabolic pathway can respond to regulatory signals such as intracellular allosteric activators or inhibitors. Intercellular signaling provides for the integration of metabolism. The primary route of this communication is chemical signaling (for example, by hormones or neurotransmitters). Second messenger molecules transduce a chemical signal (hormone or neurotransmitter binding) to appropriate intracellular responders. Adenylyl cyclase (AC) is a cell membrane enzyme that synthesizes cyclic adenosine monophosphate (cAMP) in response to chemical signals, such as the hormones glucagon and epinephrine. Following binding of a hormone to its cell-surface G protein–coupled receptor, a guanosine triphosphate–dependent regulatory protein (G protein) is activated that,


Biochemistry_Lippinco

Biochemistry_Lippinco. hormones glucagon and epinephrine. Following binding of a hormone to its cell-surface G protein–coupled receptor, a guanosine triphosphate–dependent regulatory protein (G protein) is activated that, in turn, activates AC. The cAMP produced activates protein kinase A, which phosphorylates a variety of enzymes, causing their activation or deactivation. Phosphorylation is reversed by phosphatases. Aerobic glycolysis, in which pyruvate is the end product, occurs in cells with mitochondria and an adequate supply of oxygen ([O2],


Biochemistry_Lippinco

Biochemistry_Lippinco. Fig. 8.25). Anaerobic glycolysis, in which lactic acid is the end product, occurs in cells that lack mitochondria and in cells deprived of sufficient O2. Glucose is passively transported across membranes by 1 of 14 glucose transporter (GLUT) isoforms. GLUT-1 is abundant in RBC and the brain, GLUT-4 (which is insulin dependent) in muscle and adipose tissue, and GLUT-2 in the liver, kidneys, and pancreatic β cells. The oxidation of glucose to pyruvate (glycolysis, see Fig. 8.25) occurs through an energy-investment phase in which phosphorylated intermediates are synthesized at the expense of ATP and an energy-generation phase in which ATP is produced by substrate-level phosphorylation. In the energy-investment phase, glucose is phosphorylated by hexokinase (found in most tissues) or glucokinase (a hexokinase found in liver cells and pancreatic β cells). Hexokinase has a high affinity (low Km) and a low maximal velocity (Vmax) for glucose and is inhibited by glucose 6-phosphate.


Biochemistry_Lippinco

Biochemistry_Lippinco. glucokinase (a hexokinase found in liver cells and pancreatic β cells). Hexokinase has a high affinity (low Km) and a low maximal velocity (Vmax) for glucose and is inhibited by glucose 6-phosphate. Glucokinase has a high Km and a high Vmax for glucose. It is regulated indirectly by fructose 6-phosphate (inhibits) and glucose (activates) via glucokinase regulatory protein. Glucose 6-phosphate is isomerized to fructose 6phosphate, which is phosphorylated to fructose 1,6-bisphosphate by phosphofructokinase-1 (PFK-1). This enzyme is allosterically inhibited by ATP and citrate and activated by AMP. Fructose 2,6-bisphosphate, whose synthesis by bifunctional phosphofructokinase-2 (PFK-2) is increased in the liver by insulin and decreased by glucagon, is the most potent allosteric activator of PFK-1. A total of two ATP are used during this phase of glycolysis. Fructose 1,6-bisphosphate is cleaved to form two trioses that are further metabolized by the glycolytic pathway, forming pyruvate.


Biochemistry_Lippinco

Biochemistry_Lippinco. PFK-1. A total of two ATP are used during this phase of glycolysis. Fructose 1,6-bisphosphate is cleaved to form two trioses that are further metabolized by the glycolytic pathway, forming pyruvate. During this phase, four ATP and two nicotinamide adenine dinucleotide (NADH) are produced per glucose molecule. The final step in pyruvate synthesis from phosphoenolpyruvate is catalyzed by pyruvate kinase (PK). This enzyme is allosterically activated by fructose 1,6-bisphosphate, and the hepatic isozyme is inhibited covalently by glucagon via the cAMP pathway. PK deficiency accounts for the majority of all inherited defects in glycolytic enzymes. Effects are restricted to RBC and present as mild-tosevere chronic, nonspherocytic hemolytic anemia. Glycolytic gene transcription is enhanced by insulin and glucose. In anaerobic glycolysis, NADH is reoxidized to NAD+ by the reduction of pyruvate to lactate via lactate dehydrogenase. This occurs in cells such as RBC that lack mitochondria and in


Biochemistry_Lippinco

Biochemistry_Lippinco. and glucose. In anaerobic glycolysis, NADH is reoxidized to NAD+ by the reduction of pyruvate to lactate via lactate dehydrogenase. This occurs in cells such as RBC that lack mitochondria and in tissues such as exercising muscle, where production of NADH exceeds the oxidative capacity of the respiratory chain. Elevated concentrations of lactate in the plasma (lactic acidosis) occur with circulatory system collapse or shock. Pyruvate also can be 1) oxidatively decarboxylated to acetyl CoA by pyruvate dehydrogenase, 2) carboxylated to oxaloacetate (a TCA cycle intermediate) by pyruvate carboxylase, or 3) reduced to ethanol by microbial pyruvate decarboxylase.


Biochemistry_Lippinco

Biochemistry_Lippinco. Choose the ONE best answer. .1. Which of the following best describes the activity level and phosphorylation state of the listed hepatic enzymes in an individual who consumed a carbohydrate-rich meal about an hour ago? PFK-1 = phosphofructokinase1; PFK-2 = phosphofructokinase-2; P = phosphorylated. Correct answer = C. Immediately following a meal, blood glucose levels and hepatic uptake of glucose increase. The glucose is phosphorylated to glucose 6-phosphate and used in glycolysis. In response to the rise in blood glucose, the insulin/glucagon ratio increases. As a result, the kinase domain of PFK-2 is dephosphorylated and active. Its product, fructose 2,6-bisphosphate, allosterically activates PFK-1. (PFK-1 is not covalently regulated.) Active PFK-1 produces fructose 1,6-bisphosphate that is a feedforward activator of pyruvate kinase. Hepatic pyruvate kinase is covalently regulated, and the rise in insulin favors dephosphorylation and activation.


Biochemistry_Lippinco

Biochemistry_Lippinco. .2. Which of the following statements is true for anabolic pathways only? A. Their irreversible (nonequilibrium) reactions are regulated. B. They are called cycles if they regenerate an intermediate. C. They are convergent and generate a few simple products. D. They are synthetic and require energy. E. They typically require oxidized coenzymes. Correct answer = D. Anabolic processes are synthetic and energy requiring (endergonic). Statements A and B apply to both anabolic and catabolic processes, whereas C and E apply only to catabolic processes. .3. Compared with the resting state, vigorously contracting skeletal muscle shows: A. decreased AMP/ATP ratio. B. decreased levels of fructose 2,6-bisphosphate. C. decreased NADH/NAD+ ratio. D. increased oxygen availability. E. increased reduction of pyruvate to lactate.


Biochemistry_Lippinco

Biochemistry_Lippinco. A. decreased AMP/ATP ratio. B. decreased levels of fructose 2,6-bisphosphate. C. decreased NADH/NAD+ ratio. D. increased oxygen availability. E. increased reduction of pyruvate to lactate. Correct answer = E. Vigorously contracting skeletal muscle shows an increase in the reduction of pyruvate to lactate compared with resting muscle. The levels of reduced nicotinamide adenine dinucleotide (NADH) increase and exceed the oxidative capacity of the electron transport chain. Consequently, the levels of adenosine monophosphate (AMP) increase. The concentration of fructose 2,6-bisphosphate is not a key regulatory factor in skeletal muscle. .4. Glucose uptake by: A. brain cells is through energy-requiring (active) transport. B. intestinal mucosal cells requires insulin. C. liver cells is through facilitated diffusion involving a glucose transporter. D. most cells is through simple diffusion up a concentration gradient.


Biochemistry_Lippinco

Biochemistry_Lippinco. C. liver cells is through facilitated diffusion involving a glucose transporter. D. most cells is through simple diffusion up a concentration gradient. Correct answer = C. Glucose uptake in the liver, brain, muscle, and adipose tissue is down a concentration gradient, and the diffusion is facilitated by tissue-specific glucose transporters (GLUT). In adipose and muscle tissues, insulin is required for glucose uptake. Moving glucose against a concentration gradient requires energy and is seen with the sodium-dependent glucose cotransporter 1 (SGLT1) of intestinal mucosal cells. .5. Given that the Km of glucokinase for glucose is 10 mM, whereas that of hexokinase is 0.1 mM, which isozyme will more closely approach Vmax at the normal blood glucose concentration of 5 mM?


Biochemistry_Lippinco

Biochemistry_Lippinco. .5. Given that the Km of glucokinase for glucose is 10 mM, whereas that of hexokinase is 0.1 mM, which isozyme will more closely approach Vmax at the normal blood glucose concentration of 5 mM? Correct answer = Hexokinase. Km (Michaelis constant) is that substrate concentration that gives one half Vmax (maximal velocity). When blood glucose concentration is 5 mM, hexokinase (Km = 0.1 mM) will be saturated, but glucokinase (Km = 10 mM) will not. .6. In patients with whooping cough, Gαi is inhibited. How does this lead to a rise in cyclic adenosine monophosphate (cAMP)? G proteins of the Gαi type inhibit adenylyl cyclase (AC) when their associated G protein–coupled receptor is bound by ligand. If Gαi is inhibited by pertussis toxin, AC production of cAMP is inappropriately activated. For additional ancillary materials related to this chapter, please visit thePoint. I. CYCLE OVERVIEW


Biochemistry_Lippinco

Biochemistry_Lippinco. I. CYCLE OVERVIEW The tricarboxylic acid cycle ([TCA cycle] also called the citric acid cycle, or the Krebs cycle) plays several roles in metabolism. It is the final pathway where the oxidative catabolism of carbohydrates, amino acids, and fatty acids converge, their carbon skeletons being converted to carbon dioxide (CO2), as shown in cycle. The TCA cycle is an aerobic pathway, because oxygen (O2) is required as the final electron acceptor. Reactions such as the catabolism of some amino acids generate intermediates of the cycle and are called anaplerotic (from the Greek for “filling up”) reactions. The TCA cycle also provides intermediates for a number of important anabolic reactions, such as glucose formation from the carbon skeletons of some amino acids and the synthesis of some amino acids (see p. 267) and heme (see p. 278). Therefore, this cycle should not be viewed as a closed system but, instead, as an open one with compounds entering and leaving as required.


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Biochemistry_Lippinco. II. CYCLE REACTIONS In the TCA cycle, oxaloacetate (OAA) is first condensed with an acetyl group from acetyl coenzyme A (CoA) and then is regenerated as the cycle is completed (see Fig. 9.1). Two carbons enter the cycle as acetyl CoA and two leave as CO2. Therefore, the entry of one acetyl CoA into one round of the TCA cycle does not lead to the net production or consumption of intermediates. A. Acetyl CoA production