Biochemistry, 4th Edition P60 ppt

18.5 What Are the Metabolic Fates of NADH and Pyruvate Produced in Glycolysis? 553 process. Pyruvate is decarboxylated to acetaldehyde by pyruvate decarboxylase in an essentially irreversible reaction. Thiamine pyrophosphate (see page 568) is a required cofactor for this enzyme. The second step, the reduction of acetaldehyde to ethanol by NADH, is catalyzed by alcohol dehydrogenase (Figure 18.21). At pH 7, the reaction equilibrium strongly favors ethanol. The end products of alcoholic fermentation are thus ethanol and carbon dioxide. Alcoholic fermentations are the basis for the brew- ing of beers and the fermentation of grape sugar in wine making. Lactate produced by anaerobic microorganisms during lactic acid fermentation is responsible for the taste of sour milk and for the characteristic taste and fragrance of sauerkraut, which in reality is fermented cabbage. Lactate Accumulates Under Anaerobic Conditions in Animal Tissues In animal tissues experiencing anaerobic conditions, pyruvate is reduced to lactate. Pyruvate reduction occurs in tissues that normally experience minimal access to blood flow (for example, the cornea of the eye) and also in rapidly contracting skeletal muscle. When skeletal muscles are exercised strenuously, the available tis- sue oxygen is consumed and the pyruvate generated by glycolysis can no longer be oxidized in the TCA cycle. Instead, excess pyruvate is reduced to lactate by lactate dehydrogenase (Figure 18.21). The rate of anaerobic glycolysis in skeletal muscle can increase up to 2000-fold almost instantaneously, for example, to support the in- tense demands of a sprinting animal. Large amounts of ATP are generated rapidly, at the expense of lactate accumulation. In anaerobic muscle tissue, lactate repre- sents the end of glycolysis. Anyone who exercises to the point of depleting available muscle oxygen stores knows the cramps and muscle fatigue associated with the buildup of lactic acid in the muscle. Most of this lactate must be carried out of the muscle by the blood and transported to the liver, where it can be resynthesized into glucose in gluconeogenesis. Moreover, because glycolysis generates only a fraction of the total energy available from the breakdown of glucose (the rest is generated by the TCA cycle and oxidative phosphorylation), the onset of anaerobic conditions in skeletal muscle also means a reduction in the energy available from the break- down of glucose. C + H C OH CHO CH 2 OPO 3 2– HPO 4 2– H C OH C CH 2 OPO 3 2– OPO 3 2– O H + CH 3 CHO CH 3 C COO – O Pyruvate CH 3 CH 2 OH H + OH CHO CH 2 OPO 3 2– HPO 4 2– H C OH C CH 2 OPO 3 2– OPO 3 2– O H + CH 3 C COO – COO – O CH 3 C OH H G3PDH CO 2 NAD + NADH NAD + NADH D-Glyceraldehyde- 3-phosphate 1,3-BPG AcetaldehydeEthanol D-Glyceraldehyde- 3-phosphate 1,3-BPG Lactate Pyruvate Lactate dehydrogenase (a) Alcoholic fermentation (b) Lactic acid fermentation G3PDH Alcohol dehydrogenase FIGURE 18.21 (a) Pyruvate reduction to ethanol in yeast provides a means for regenerating NAD ϩ consumed in the glyceraldehyde-3-P dehydrogenase reaction. (b) In oxygen-depleted muscle, NAD ϩ is regenerated in the lactate dehydrogenase reaction. 554 Chapter 18 Glycolysis 18.6 How Do Cells Regulate Glycolysis? The elegance of nature’s design for the glycolytic pathway may be appreciated through an examination of Figure 18.22. The standard-state free energy changes for the 10 re- actions of glycolysis and the lactate dehydrogenase reaction (Figure 18.22a) are vari- ously positive and negative and, taken together, offer little insight into the coupling that occurs in the cellular milieu. On the other hand, the values of ⌬G under cellular conditions (Figure 18.22b) fall into two distinct classes. For reactions 2 and 4 through 9, ⌬G is very close to zero, meaning these reactions operate essentially at equilibrium. Small changes in the concentrations of reactants and products could “push” any of these reactions either forward or backward. By contrast, the hexokinase, phospho- fructokinase, and pyruvate kinase reactions all exhibit large negative ⌬G values under cellular conditions. These reactions are thus the sites of glycolytic regulation. When these three enzymes are active, glycolysis proceeds and glucose is readily metabolized to pyruvate or lactate. Inhibition of the three key enzymes by allosteric effectors brings glycolysis to a halt. When we consider gluconeogenesis—the biosynthesis of glucose— in Chapter 22, we will see that different enzymes are used to carry out reactions 1, 3, and 10 in reverse, effecting the net synthesis of glucose. The maintenance of reactions 2 and 4 through 9 at or near equilibrium permits these reactions (and their respective enzymes!) to operate effectively in either the forward or reverse direction. 18.7 Are Substrates Other Than Glucose Used in Glycolysis? The glycolytic pathway described in this chapter begins with the breakdown of glucose, but other sugars, both simple and complex, can enter the cycle if they can be converted by appropriate enzymes to one of the intermediates of glycoly- sis. Figure 18.23 shows the routes by which several simple metabolites can enter (a) ΔG at standard state (ΔG°') 40 30 20 10 0 –10 –20 –30 –40 Free energy, kJ/mol 0 Steps of glycolysis 1234567891011 40 30 20 10 0 –10 –20 –30 –40 Free energy, kJ/mol 0 Ste p s of glycolysis 1234567891011 (b) ΔG in erythrocytes (ΔG) FIGURE 18.22 A comparison of free energy changes for the reactions of glycolysis (step 1 ϭ hexokinase) under (a) standard-state conditions and (b) actual intracellular conditions in erythrocytes.The values of ⌬G°Ј provide little insight into the actual free energy changes that occur in glycolysis. On the other hand, under intracellu- lar conditions, seven of the glycolytic reactions operate near equilibrium (with ⌬G near zero).The driving force for glycolysis lies in the hexokinase (1), phosphofructo- kinase (3), and pyruvate kinase (10) reactions. The lactate dehydrogenase (step 11) reaction also exhibits a large negative ⌬G under cellular conditions. G-6-P F-6-P FBP 2 Pyruvate Mannose-6-P Mannose Galactose Galactose-1-PUDP-Gal UDP-Glucose Glucose-1-P Fructose Fructose-1-P D-Glyceraldehyde Aldolase Fructokinase Triose kinase Glucose 3-PG 2-PG PEP DHAP G-3-P BPG FIGURE 18.23 Mannose, galactose, fructose, and other simple metabolites can enter the glycolytic pathway. 18.7 Are Substrates Other Than Glucose Used in Glycolysis? 555 the glycolytic pathway. Fructose, for example, which is produced by breakdown of sucrose, may participate in glycolysis by at least two different routes. In the liver, fructose is phosphorylated at C-1 by the enzyme fructokinase: D-Fructose ϩ ATP 4Ϫ ⎯⎯→D-fructose-1-phosphate 2Ϫ ϩ ADP 3Ϫ ϩ H ϩ (18.9) Subsequent action by fructose-1-phosphate aldolase cleaves fructose-1-P in a man- ner like the fructose bisphosphate aldolase reaction to produce dihydroxyacetone phosphate and D-glyceraldehyde: D-Fructose-1-P 2Ϫ ⎯⎯→D-glyceraldehyde ϩ dihydroxyacetone phosphate 2Ϫ (18.10) Dihydroxyacetone phosphate is of course an intermediate in glycolysis. D-Glycer- aldehyde can be phosphorylated by triose kinase in the presence of ATP to form D-glyceraldehyde-3-phosphate, another glycolytic intermediate. In the kidney and in muscle tissues, fructose is readily phosphorylated by hex- okinase, which, as pointed out previously, can utilize several different hexose sub- strates. The free energy of hydrolysis of ATP drives the reaction forward: D-Fructose ϩ ATP 4Ϫ ⎯⎯→D-fructose-6-phosphate 2Ϫ ϩ ADP 3Ϫ ϩ H ϩ (18.11) HUMAN BIOCHEMISTRY Tumor Diagnosis Using Positron Emission Tomography (PET) More than 70 years ago, Otto Warburg at the Kaiser Wilhelm In- stitute of Biology in Germany demonstrated that most animal and human tumors displayed a very high rate of glycolysis compared to that of normal tissue. This observation from long ago is the ba- sis of a modern diagnostic method for tumor detection called positron emission tomography, or PET. PET uses molecular probes that contain a neutron-deficient, radioactive element such as carbon-11 or fluorine-18. An example is 2-[ 18 F]fluoro-2-deoxy- glucose (FDG), a molecular mimic of glucose. The 18 F nucleus is unstable and spontaneously decays by emission of a positron (an antimatter* particle) from a proton, thus converting a proton to a neutron and transforming the 18 F to 18 O. The emitted positron typically travels a short distance (less than a millimeter) and col- lides with an electron, annihilating both particles and creating a pair of high-energy photons—gamma rays. Detection of the gamma rays with special cameras can be used to construct three- dimensional models of the location of the radiolabeled molecu- lar probe in the tissue of interest. FDG is taken up by human cells and converted by hexokinase to 2-[ 18 F]fluoro-2-deoxy-glucose-6-phosphate in the first step of glycolysis. Cells of a human brain, for example, accumulate FDG in direct proportion to the amount of glycolysis occuring in those cells. Tumors can be identified in PET scans as sites of unusually high FDG accumulation. HO OH 18 F HOH (a) (b) CH 2 OH 2-[ 18 F]Fluoro-2-deoxyglucose Electron in tissue 511 kev Photon 511 kev Photon 18 F 18 O Emitted positron O e + e – NIH/Science Source/Photo Researchers, Inc. (c) PET image of human brain following administration of 18 FDG. Red area indicates a large malignant tumor. *The existence of antimatter in the form of positrons was first postulated by Robert Oppenheimer, the father of the atomic bomb. 556 Chapter 18 Glycolysis Fructose-6-phosphate generated in this way enters the glycolytic pathway directly in step 3, the second priming reaction. This is the principal means for channeling fructose into glycolysis in adipose tissue, which contains high levels of fructose. Mannose Enters Glycolysis in Two Steps Another simple sugar that enters glycolysis at the same point as fructose is mannose, which occurs in many glycoproteins, glycolipids, and polysaccharides (see Chapter 7). Mannose is also phosphorylated from ATP by hexokinase, and the mannose-6-phosphate thus produced is converted to fructose-6-phosphate by phosphomannoisomerase. D-Mannose ϩ ATP 4Ϫ ⎯→ D-mannose-6-phosphate 2Ϫ ϩ ADP 3Ϫ ϩ H ϩ (18.12) D-Mannose-6-phosphate 2Ϫ ⎯⎯→D-fructose-6-phosphate 2Ϫ (18.13) Galactose Enters Glycolysis Via the Leloir Pathway A somewhat more complicated route into glycolysis is followed by galactose, an- other simple hexose sugar. The process, called the Leloir pathway after Luis Leloir, its discoverer, begins with phosphorylation from ATP at the C-1 position by galactokinase: D-Galactose ϩ ATP 4Ϫ ⎯⎯→D-galactose-1-phosphate 2Ϫ ϩ ADP 3Ϫ ϩ H ϩ (18.14) Galactose-1-phosphate is then converted into UDP-galactose (a sugar nucleotide) by galactose-1-phosphate uridylyltransferase (Figure 18.24), with concurrent pro- duction of glucose-1-phosphate and consumption of a molecule of UDP-glucose. The uridylyltransferase reaction (Figure 18.25) proceeds via a “ping-pong” mech- anism (see Chapter 13, page 406) with a covalent enzyme-UMP intermediate. The glucose-1-phosphate produced by the transferase reaction is a substrate for the phosphoglucomutase reaction (Figure 18.24), which produces glucose-6- phosphate, a glycolytic substrate. The other transferase product, UDP-galactose, is converted to UDP-glucose by UDP-glucose-4-epimerase. The combined action of the uridylyltransferase and epimerase thus produces glucose-1-P from galactose- 1-P, with regeneration of UDP-glucose. A rare hereditary condition known as galactosemia involves defects in galactose- 1-P uridylyltransferase that render the enzyme inactive. Toxic levels of galactose accumulate in afflicted individuals, causing cataracts and permanent neurological disorders. These problems can be prevented by removing galactose and lactose from the diet. In adults, the toxicity of galactose appears to be less severe, due in part to the metabolism of galactose-1-P by UDP-glucose pyrophosphorylase, which Galactose-1- uridylyltransferase UDP-Galactose- 4-epimerase Phosphoglucomutase Galactokinase Galactose P Galactose-1- P UDP-Glucose UDP-Galactose P Glucose-1- P Glucose-6- ATP ADP FIGURE 18.24 Galactose metabolism via the Leloir pathway. 18.7 Are Substrates Other Than Glucose Used in Glycolysis? 557 apparently can accept galactose-1-P in place of glucose-1-P (Figure 18.26). The levels of this enzyme may increase in galactosemic individuals in order to accom- modate the metabolism of galactose. An Enzyme Deficiency Causes Lactose Intolerance A much more common metabolic disorder, lactose intolerance, occurs com- monly in most parts of the world (notable exceptions being some parts of Africa and northern Europe). Lactose intolerance is an inability to digest lactose be- cause of the absence of the enzyme lactase in the intestines of adults. The symp- toms of this disorder, which include diarrhea and general discomfort, can be re- lieved by eliminating milk from the diet. Glycerol Can Also Enter Glycolysis Glycerol is the last important simple substance whose ability to enter the glycolytic pathway must be considered. This metabolite, which is produced in substantial amounts by the decomposition of triacylglycerols (see Chapter 23), can be converted O O P O – O P O – HO HOH CH 2 OH O OOUridine + O – O P O – H HOH CH 2 OH O O O – O P O – HO HOH CH 2 OH O O O O P O – O P O – HO HOH CH 2 OH O OOUridine + OH HO OH OH OH UDP-glucose ␣-D-Galactose-1-P ␣- D-Glucose-1-P UDP-galactose FIGURE 18.25 The galactose-1-phosphate uridylyl- transferase reaction involves a “ping-pong”kinetic mechanism. O – O P O – H HOH CH 2 OH O O + CH 2 P O O HN HO HOH CH 2 OH O O HO OH O OHHO H N OP OPO O O – O O – O O – – O CH 2 O OHHO H P OPO O O – O O – OH O HN N O – OP O – PO O O – O O – + ␣-D-Galactose-1-P UDP-galactose (UDP-Gal) UTP Pyrophosphate FIGURE 18.26 The UDP-glucose pyrophosphorylase reaction also works with galactose-1-P. 558 Chapter 18 Glycolysis to glycerol-3-phosphate by the action of glycerol kinase and then oxidized to dihy- droxyacetone phosphate by the action of glycerol phosphate dehydrogenase, with NAD ϩ as the required coenzyme. The dihydroxyacetone phosphate thereby produced enters the glycolytic pathway as a substrate for triose phosphate isomerase. HUMAN BIOCHEMISTRY Lactose—From Mother’s Milk to Yogurt—and Lactose Intolerance Lactose is an interesting sugar in many ways. In placental mam- mals, it is synthesized only in the mammary gland, and then only during late pregnancy and lactation. The synthesis is carried out by lactose synthase, a dimeric complex of two proteins: galactosyl transferase and ␣-lactalbumin. Galactosyl transferase is present in all human cells, and it is normally involved in incorporation of galactose into glycoproteins. In late pregnancy, the pituitary gland in the brain releases a protein hormone, prolactin, which triggers production of ␣-lactalbumin by certain cells in the breast. ␣-Lactalbumin, a 123-residue protein, associates with galactosyl transferase to form lactose synthase, which catalyzes the reaction: UDP-galactose ϩ glucose⎯⎯→lactose ϩ UDP Lactose breakdown by lactase in the small intestine provides newborn mammals with essential galactose for many purposes, including the synthesis of gangliosides in the developing brain. Lactase is a ␤-galactosidase that cleaves lactose to yield galactose and glucose—in fact, the only human enzyme that can cleave a ␤-glycosidic linkage: Lactase is an inducible enzyme in mammals, and it appears in the fetus only during the late stages of gestation. Lactase activity peaks shortly after birth, but by the age of 3 to 5 years, it declines to a low level in nearly all human children. Low levels of lactase make many adults lactose intolerant. Lactose intolerance occurs commonly in most parts of the world (with the notable exception of some parts of Africa and northern Europe; see table). The symptoms of lactose in- tolerance, including diarrhea and general discomfort, can be relieved by eliminatin g milk from the diet. Alternatively, products containing ␤-galactosidase are available commercially. Certain bacteria, including several species of Lactobacillus, thrive on the lactose in milk and carry out lactic acid fermentation, con- verting lactose to lactate via glycolysis. This is the basis of produc- tion of yogurt, which is now popular in the Western world but of Turkish origin. Other cultures also produce yogurtlike foods. No- madic Tatars in Siberia and Mongolia used camel milk to make koumiss, which was used for medicinal purposes. In the Caucasus, kefir is made much like yogurt, except that the starter culture con- tains (in addition to Lactobacillus) Streptococcus lactis and yeast, which convert some of the glucose to ethanol and CO 2 , producing an effervescent and slightly intoxicating brew. HO OH OH HOH CH 2 OH O HO OH OH HOH + CH 2 OH O OH OH HOH CH 2 OH O O HO OH OH Lactose Galactose Glucose CH 2 OH O Lactase ᮡ Breakdown of lactose to galactose and glucose by lactase. Portions adapted from Hill, R., and Brew, K., 1975. Lactose synthetase. Ad- vances in Enzymology 43:411–485; and Bloch, K., 1994. Blondes in Venetian Paintings, the Nine-Banded Armadillo, and Other Essays in Biochemistry. New Haven, CT: Yale University Press. Country Lactase Persistence (%) Sweden 99 Denmark 97 United Kingdom (Scotland) 95 Germany 88 Australia 82 United States (Iowa) 81 Spain 72 France 58 India 36 Japan 10 China (Singapore) 0 Adapted from Bloch, K.,1994. Blondes in Venetian Paintings, the Nine-Banded Armadillo, and Other Essays in Biochemistry. New Haven, CT:Yale University Press. Percentage of Population with Lactase Persistence Glycerol kinase reaction + CH 2 OH HOCH CH 2 OH Mg 2+ CH 2 OH HOCH CH 2 OPO 3 2– + ATP ADP Glycerol sn-Glycerol-3-phosphate 18.8 How Do Cells Respond to Hypoxic Stress? 559 18.8 How Do Cells Respond to Hypoxic Stress? Glycolysis is an anaerobic pathway—it does not require oxygen. But as noted in Fig- ure 18.1, operation of the TCA cycle (the subject of Chapter 19) depends on oxy- gen, so it is aerobic. When oxygen is abundant, cells prefer aerobic metabolism, which yields more energy per glucose consumed. However, as Louis Pasteur first showed, when oxygen is limited, cells adapt to make the most of glycolysis, the less energetic, anaerobic alternative. In mammalian tissues, hypoxia (oxygen limitation) can cause changes in gene expression that result in increased angiogenesis (the growth of new blood vessels), increased synthesis of red blood cells, and increased levels of some glycolytic enzymes (and thus a higher rate of glycolysis). What is the molecular basis for the increased expression of glycolytic enzymes? One of the triggers for this expression is a DNA-binding protein called hypoxia inducible factor (HIF). HIF is a heterodimer of a constitutive nuclear subunit (HIF-1␤) and an inducible ␣-subunit. Both subunits are basic helix-loop-helix tran- scription factors that bind to hypoxia-inducible genes, and both subunits exist as a series of isoforms (for example, HIF-1␣, HIF-2␣, and HIF-3␣). HIF-␣ subunit regu- lation is a multistep process that includes gene splicing, phosphorylation, acetyla- tion, and hydroxylation. HIF-1␣ is the best-studied HIF-␣ isoform. When oxygen is plentiful, HIF-1␣ is hydroxylated by oxygen-dependent prolyl hydroxylases (PHDs) at Pro 402 and Pro 564 . These hydroxylations ensure its binding to ubiquitin E3 ligase, which leads to rapid proteolysis by the 26S proteasome (see Chapter 31). HIF-1␣ binding to the ligase is also promoted by acetylation of Lys 532 by the ARD1 acetyl- transferase. In addition, the presence of oxygen induces the hydroxylation of HIF-1␣ Asn 803 by the hydroxylase factor–inhibiting HIF (FIH-1). Hydroxylation in- hibits the transcription activity of HIF-1␣ by preventing its interaction with the acti- vator p300. Figure 18.27 shows the structure of FIH bound to a fragment of HIF-1␣. Because PHDs and FIH-1 both are oxygen-dependent, lowering oxygen concen- tration means that HIF-1␣ avoids degradation and is available to promote gene tran- scription (Figure 18.28). Phosphorylation of HIF-1␣ by a protein kinase promotes Glycerol-P dehydrogenase reaction H + + CH 2 OH HOCH CH 2 OPO 3 2– CH 2 OH C CH 2 OPO 3 2– + O + NAD + NADH sn-Glycerol-3-phosphate Dihydroxyacetone phosphate FIGURE 18.27 FIH (green) bound to HIF. Oxygen levels Pro 564 HO Pro 564 HO Degradation Transcription Cofactors O 2 Fe 2 + Pro 564 HIF-1␣ Proteasome HIF-1␣ HIF-1␣ HIF-1␣ Cofactor PHD HRE HIF-1␤ VHL FIGURE 18.28 The HIF transcription factor is composed of two subunits: a ubiquitous HIF-1␤ subunit and a hypoxia-responsive HIF-1␣ subunit.In response to hypoxia, inactivation of the PHDs allows HIF-1␣ stabiliza- tion, dimerization with HIF-1␤, binding of the dimer to the hypoxia responsive element (HRE) of HIF target genes, and activation of the transcription of these genes. VHL is the von Hippel Lindau subunit of the ubiquitin E3 ligase that targets proteins for proteasome degrada- tion. (Adapted from North, S., Moenner, M.,and Bikfalvi,A., 2006. Recent developments in the regulation of the angiogenic switch by cellular switch factors in tumors. Cancer Letters 218:1–14.) 560 Chapter 18 Glycolysis binding of HIF-1␣ to HIF-1␤, which enhances transcription. HIF-1␣–HIF-1␤ dimers bind to hypoxia responsive elements (HREs), activating transcription of HRE- regulated genes, including genes for glycolytic enzymes. Pasteur observed more than 100 years ago that fermentation amounted to “life without air.” The “Pasteur effect” depends on HIF-mediated activation of the genes encoding glycolytic enzymes in the absence of oxygen. The linking of glycolytic activity to oxygen level is the result of an exquisite dance of oxygen-sensitive enzymes with proteins, which undergo covalent modifications that control protein–protein and protein–DNA interactions, a dance that Pasteur could hardly have anticipated. SUMMARY Nearly every living cell carries out a catabolic process known as glycolysis— the stepwise degradation of glucose (and other simple sugars). Glycolysis is a paradigm of metabolic pathways. Localized in the cytosol of cells, it is basically an anaerobic process; its principal steps occur with no require- ment for oxygen. 18.1 What Are the Essential Features of Glycolysis? Glycolysis con- sists of two phases. In the first phase, a series of five reactions, glucose is broken down to two molecules of glyceraldehyde-3-phosphate. In the second phase, five subsequent reactions convert these two molecules of glyceraldehyde-3-phosphate into two molecules of pyruvate. Phase 1 consumes two molecules of ATP. The later stages of glycolysis result in the production of four molecules of ATP. The net is 4 Ϫ 2 ϭ 2 molecules of ATP produced per molecule of glucose. 18.2 Why Are Coupled Reactions Important in Glycolysis? Coupled reactions permit the energy of glycolysis to be used for generation of ATP. Conversion of glucose to pyruvate in glycolysis drives the produc- tion of two molecules of ATP. 18.3 What Are the Chemical Principles and Features of the First Phase of Glycolysis? In the first phase of glycolysis, glucose is converted into two molecules of glyceraldehyde-3-phosphate. First, glucose is phos- phorylated to glucose-6-P, which is isomerized to fructose-6-P. Another phosphorylation and then cleavage yields two 3-carbon intermediates. One of these is glyceraldehyde-3-P, and the other, dihydroxyacetone-P, is converted to glyceraldehyde-3-P. Energy released from this high- energy molecule in the second phase of glycolysis is then used to syn- thesize ATP. 18.4 What Are the Chemical Principles and Features of the Second Phase of Glycolysis? The second half of the glycolytic pathway involves the re- actions that convert the metabolic energy in the glucose molecule into ATP. Phase 2 starts with the oxidation of glyceraldehyde-3-phosphate, a re- action with a large enough energy “kick” to produce a high-energy phos- phate, namely, 1,3-bisphosphoglycerate. Phosphoryl transfer from 1,3-BPG to ADP to make ATP is highly favorable. The product, 3-phosphoglycerate, is converted via several steps to phosphoenolpyruvate (PEP), another high-energy phosphate. PEP readily transfers its phosphoryl group to ADP in the pyruvate kinase reaction to make another ATP. 18.5 What Are the Metabolic Fates of NADH and Pyruvate Produced in Glycolysis? In addition to ATP, the products of glycolysis are NADH and pyruvate. Their processing depends upon other cellular pathways. NADH must be recycled to NAD ϩ , lest NAD ϩ become limiting in gly- colysis. NADH can be recycled by both aerobic and anaerobic paths, ei- ther of which results in further metabolism of pyruvate. What a given cell does with the pyruvate produced in glycolysis depends in part on the availability of oxygen. Under aerobic conditions, pyruvate can be sent into the citric acid cycle, where it is oxidized to CO 2 with the pro- duction of additional NADH (and FADH 2 ). Under aerobic conditions, the NADH produced in glycolysis and the citric acid cycle is reoxidized to NAD ϩ in the mitochondrial electron-transport chain. Under anaerobic conditions, the pyruvate produced in glycolysis is not sent to the citric acid cycle. Instead, it is reduced to ethanol in yeast; in other microorganisms and in animals, it is reduced to lactate. These processes are examples of fermentation—the production of ATP energy by reaction pathways in which organic molecules function as donors and acceptors of electrons. In either case, reduction of pyruvate provides a means of reoxidizing the NADH produced in the glyceraldehyde-3- phosphate dehydrogenase reaction of glycolysis. 18.6 How Do Cells Regulate Glycolysis? The standard-state free en- ergy changes for the 10 reactions of glycolysis are variously positive and negative and, taken together, offer little insight into the coupling that occurs in the cellular milieu. On the other hand, the values of ⌬G un- der cellular conditions fall into two distinct classes. For reactions 2 and 4 through 9, ⌬G is very close to zero, meaning these reactions operate essentially at equilibrium. Small changes in the concentrations of reac- tants and products could “push” any of these reactions either forward or backward. By contrast, the hexokinase, phosphofructokinase, and pyruvate kinase reactions all exhibit large negative ⌬G values under cel- lular conditions. These reactions are thus the sites of glycolytic regula- tion. 18.7 Are Substrates Other Than Glucose Used in Glycolysis? Fructose enters glycolysis by either of two routes. Mannose, galactose, and glyc- erol enter via reactions that are linked to the glycolytic pathway. 18.8 How Do Cells Respond to Hypoxic Stress? Glycolysis is an anaer- obic pathway, but it normally feeds pyruvate into aerobic metabolic pathways. However, when oxygen is limited, cells adapt to make the most of glycolysis. In mammalian tissues, oxygen limitation (hypoxia) can cause changes in gene expression that result in increased angio- genesis, red blood cell synthesis, and elevated levels of some glycolytic enzymes. One of the triggers for this expression is a DNA-binding pro- tein, HIF, which binds to hypoxia-inducible genes. HIF-␣ regulation is a multistep process that includes gene splicing, phosphorylation, acetyla- tion, and hydroxylation. The Pasteur effect depends on HIF-mediated activation of the genes encoding glycolytic enzymes in the absence of oxygen. PROBLEMS Preparing for an exam? Create your own study path for this chapter at www.cengage.com/login. 1. List the reactions of glycolysis that a. are energy consuming (under standard-state conditions). b. are energy yielding (under standard-state conditions). c. consume ATP. d. yield ATP. e. are strongly influenced by changes in concentration of substrate and product because of their molecularity. f. are at or near equilibrium in the erythrocyte (see Table 18.2). 2. Determine the anticipated location in pyruvate of labeled carbons if glucose molecules labeled (in separate experiments) with 14 C at each position of the carbon skeleton proceed through the glycolytic pathway. 3. In an erythrocyte undergoing glycolysis, what would be the effect of a sudden increase in the concentration of a. ATP? b. AMP? c. fructose-1,6-bisphosphate? d. fructose-2,6-bisphosphate? e. citrate? f. glucose-6-phosphate? 4. Discuss the cycling of NADH and NAD ϩ in glycolysis and the related fermentation reactions. 5. For each of the following reactions, name the enzyme that carries out this reaction in glycolysis and write a suitable mechanism for the reaction. 6. Write the reactions that permit galactose to be utilized in glycolysis. Write a suitable mechanism for one of these reactions. 7. (Integrates with Chapters 4 and 14.) How might iodoacetic acid affect the glyceraldehyde-3-phosphate dehydrogenase reaction in glycolysis? Justify your answer. 8. If 32 P-labeled inorganic phosphate were introduced to erythrocytes undergoing glycolysis, would you expect to detect 32 P in glycolytic intermediates? If so, describe the relevant reactions and the 32 P incorporation you would observe. 9. Sucrose can enter glycolysis by either of two routes: Sucrose phosphorylase: Sucrose ϩ P i 34 fructose ϩ glucose-1-phosphate Invertase: Sucrose ϩ H 2 O 34 fructose ϩ glucose Would either of these reactions offer an advantage over the other in the preparation of hexoses for entry into glycolysis? 10. What would be the consequences of a Mg 2ϩ ion deficiency for the reactions of glycolysis? CO HOCH HCOH HCOH CH 2 OPO 3 2Ϫ CH 2 OPO 3 2Ϫ CO ϩ CH 2 OPO 3 2Ϫ CH 2 OH CHO HCOH CH 2 OPO 3 2Ϫ CHO HCOH CH 2 OPO 3 2Ϫ C HCOH CH 2 OPO 3 2Ϫ OPO 3 2Ϫ O 11. (Integrates with Chapter 3.) Triose phosphate isomerase catalyzes the conversion of dihydroxyacetone-P to glyceraldehyde-3-P. The standard free energy change, ⌬G°Ј, for this reaction is ϩ7.6 kJ/mol. However, the observed free energy change (⌬G) for this reaction in erythrocytes is ϩ2.4 kJ/mol. a. Calculate the ratio of [dihydroxyacetone-P]/[glyceraldehyde-3-P] in erythrocytes from ⌬G. b. If [dihydroxyacetone-P] ϭ 0.2 mM, what is [glyceraldehyde-3-P]? 12. (Integrates with Chapter 3.) Enolase catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate ϩ H 2 O. The standard free energy change, ⌬G°Ј, for this reaction is ϩ1.8 kJ/mol. If the concentration of 2-phosphoglycerate is 0.045 mM and the concen- tration of phosphoenolpyruvate is 0.034 mM, what is ⌬G, the free energy change for the enolase reaction, under these conditions? 13. (Integrates with Chapter 3.) The standard free energy change (⌬G°Ј) for hydrolysis of phosphoenolpyruvate (PEP) is Ϫ61.9 kJ/mol. The standard free energy change (⌬G°Ј) for ATP hydrolysis is Ϫ30.5 kJ/mol. a. What is the standard free energy change for the pyruvate kinase reaction: ADP ϩ phosphoenolpyruvate⎯⎯→ ATP ϩ pyruvate b. What is the equilibrium constant for this reaction? c. Assuming the intracellular concentrations of [ATP] and [ADP] remain fixed at 8 mM and 1 mM, respectively, what will be the ratio of [pyruvate]/[phosphoenolpyruvate] when the pyruvate ki- nase reaction reaches equilibrium? 14. (Integrates with Chapter 3.) The standard free energy change (⌬G°Ј) for hydrolysis of fructose-1,6-bisphosphate (FBP) to fructose- 6-phosphate (F-6-P) and P i is Ϫ16.7 kJ/mol: FBP ϩ H 2 O⎯⎯→fructose-6-P ϩ P i The standard free energy change (⌬G°Ј) for ATP hydrolysis is Ϫ30.5 kJ/mol: ATP ϩ H 2 O⎯⎯→ADP ϩ P i a. What is the standard free energy change for the phosphofructo- kinase reaction: ATP ϩ fructose-6-P⎯⎯→ADP ϩ FBP b. What is the equilibrium constant for this reaction? c. Assuming the intracellular concentrations of [ATP] and [ADP] are maintained constant at 4 mM and 1.6 mM, respectively, in a rat liver cell, what will be the ratio of [FBP]/[fructose-6-P] when the phosphofructokinase reaction reaches equilibrium? 15. (Integrates with Chapter 3.) The standard free energy change (⌬G°Ј) for hydrolysis of 1,3-bisphosphoglycerate (1,3-BPG) to 3- phosphoglycerate (3-PG) and P i is Ϫ49.6 kJ/mol: 1,3-BPG ϩ H 2 O⎯⎯→3-PG ϩ P i The standard free energy change (⌬G°Ј) for ATP hydrolysis is Ϫ30.5 kJ/mol: ATP ϩ H 2 O⎯⎯→ADP ϩ P i a. What is the standard free energy change for the phosphoglycerate kinase reaction: ADP ϩ 1,3-BPG⎯⎯→ ATP ϩ 3-PG b. What is the equilibrium constant for this reaction? c. If the steady-state concentrations of [1,3-BPG] and [3-PG] in an erythrocyte are 1 ␮M and 120 ␮M, respectively, what will be the ratio of [ATP]/[ADP], assuming the phosphoglycerate kinase re- action is at equilibrium? Problems 561 562 Chapter 18 Glycolysis 16. The standard-state free energy change, ⌬G°Ј, for the hexokinase re- action is Ϫ16.7 kJ/mol. Use the values in Table 18.2 to calculate the value of ⌬G for this reaction in the erythrocyte at 37°C. 17. Taking into consideration the equilibrium constant for the adeny- late kinase reaction (page 542), calculate the change in concentra- tion in AMP that would occur if 8% of the ATP in an erythrocyte (red blood cell) were suddenly hydrolyzed to ADP. In addition to the concentration values in Table 18.2, it may be useful to assume that the initial concentration of AMP in erythrocytes is 5 ␮M. 18. Fructose bisphosphate aldolase in animal muscle is a class I aldolase, which forms a Schiff base intermediate between substrate (for ex- ample, fructose-1,6-bisphosphate or dihydroxyacetone phosphate) and a lysine at the active site (see Figure 18.12). The chemical evi- dence for this intermediate comes from studies with aldolase and the reducing agent sodium borohydride, NaBH 4 . Incubation of the enzyme with dihydroxyacetone phosphate and NaBH 4 inactivates the enzyme. Interestingly, no inactivation is observed if NaBH 4 is added to the enzyme in the absence of substrate. Write a mechanism that explains these observations and provides evidence for the for- mation of a Schiff base intermediate in the aldolase reaction. 19. As noted on page 556, the galactose-1-phosphate uridylyltransferase reaction proceeds via a ping-pong mechanism. Consult Chapter 13, page 406, to refresh your knowledge of ping-pong mechanisms, and draw a diagram to show how a ping-pong mechanism would pro- ceed for the uridylyltransferase. 20. Genetic defects in glycolytic enzymes can have serious conse- quences for humans. For example, defects in the gene for pyruvate kinase can result in a condition known as hemolytic anemia. Con- sult a reference to learn about hemolytic anemia, and discuss why such genetic defects lead to this condition. Preparing for the MCAT Exam 21. Regarding phosphofructokinase, which of the following statements is true: a. Low ATP stimulates the enzyme, but fructose-2,6-bisphosphate inhibits. b. High ATP stimulates the enzyme, but fructose-2,6-bisphosphate inhibits. c. High ATP stimulates the enzyme, but fructose-2,6-bisphosphate inhibits. d. The enzyme is more active at low ATP than at high, and fructose- 2,6-bisphosphate activates the enzyme. e. ATP and fructose-2,6-bisphosphate both inhibit the enzyme. 22. Based on your reading of this chapter, what would you expect to be the most immediate effect on glycolysis if the steady-state concen- tration of glucose-6-P were 8.3 mM instead of 0.083 mM ? FURTHER READING General Fothergill-Gilmore, L., 1986. The evolution of the glycolytic pathway. Trends in Biochemical Sciences 11:47–51. Kim, J-W., and Dang, C. V., 2006. Cancer’s molecular sweet tooth and the Warburg effect. Cancer Research 66:8927–8930. Sparks, S., 1997. The purpose of glycolysis. Science 277:459–460. Waddell, T. G., 1997. Optimization of glycolysis: A new look at the effi- ciency of energy coupling. Biochemical Education 25:204–205. Enzymes of Glycolysis Aleshin, A. E., Kirby, C., et al., 2000. Crystal structures of mutant monomeric hexokinase I reveal multiple ADP-binding sites and conformational changes relevant to allosteric regulation. Journal of Molecular Biology 296:1001–1015. Choi, K. H., Shi, J., et al., 2001. Snapshots of catalysis: The structure of fructose-1,6-(bis)phosphate aldolase covalently bound to the sub- strate dihydroxyacetone phosphate. Biochemistry 40:13868–13875. Didierjean, C., Corbier, C., et al., 2003. Crystal structure of two ternary complexes of phosphorylating glyceraldehyde-3-phosphate dehydro- genase from Bacillus sterothermophilus with NAD and D-glyceraldehyde 3-phosphate. Journal of Biological Chemistry 278:12968–12976. Jeffery, C. J., 1999. Moonlighting proteins. Trends in Biochemical Sciences 24:8–11. Jeffery, C. J., 2004. Molecular mechanisms for multitasking: Recent crys- tal structures of moonlighting proteins. Current Opinion in Structural Biology 14:663–668. Kim, J-W., and Dang, C. V, 2005. Multifaceted roles of glycolytic en- zymes. Trends in Biochemical Sciences 30:142–150. Lee, J. H., Chang, K. Z., et al., 2001. Crystal structure of rabbit phos- phoglucose isomerase complexed with its substrate D-fructose 6- phosphate. Biochemistry 40:7799–7805. Lolis, E., and Petsko, G., 1990. Crystallographic analysis of the complex between triosephosphate isomerase and 2-phosphoglycolate at 2.5 Å resolution: Implications for catalysis. Biochemistry 29:6619–6625. Schirmer, T., and Evans, P. R., 1999. Structural basis of the allosteric be- haviour of phosphofructokinase. Nature 343:140–145. Valentini, G., Chiarelli, L., et al., 2000. The allosteric regulation of pyru- vate kinase. Journal of Biological Chemistry 275:18145–18152. Wilson, J. E., 2003. Isozymes of mammalian hexokinase: Structure, sub- cellular localization and metabolic function. Journal of Experimental Biology 206:2049–2057. Zhang, E., Brewer, J. M., et al., 1997. Mechanism of enolase: The crystal structure of asymmetric dimer enolase-2-phospho- D-glycerate/ enolase-phosphoenopyruvate at 2.0 Å resolution. Biochemistry 36: 12526–12534. Muscle Biochemistry Green, H. J., 1997. Mechanisms of muscle fatigue in intense exercise. Journal of Sports Sciences 15:247–256. HIF-1␣ and Glycolysis Cramer, T., Yamanishi, Y., et al., 2003. HIF-1␣ is essential for myeloid cell-mediated inflammation. Cell 112:645–657. Melillo, G., 2006. Inhibiting hypoxia-inducible factor 1 for cancer ther- apy. Molecular Cancer Research 4:601–605. Melillo, G., 2007. Targeting hypoxia cell signaling for cancer therapy. Cancer and Metastasis Reviews 26:341–352. North, S., Moenner, M., et al., 2005. Recent developments in the regu- lation of the angiogenic switch by cellular stress factors in tumors. Cancer Letters 218:1–14.