S-162Chapter 14Glycolysis, Gluconeogenesis, and the Pentose Phosphate PathwayAnswerFor this reaction, GGRTln [glyceraldehyde 3-phosphate][dihydroxyace-tone phosphate]/[fructose 1,6-bisphosphate]: G23.8 kJ/mol (8.315 103kJ/mol K) (310 K) ln [(3 106)(1.6 105)/(1.4 105)]23.8 kJ/mol (2.578 kJ/mol) ln (3.43 106)23.8 kJ/mol (2.578 kJ/mol) (12.58)23.8 kJ/mol (32.4 kJ/mol) 8.6 kJ/mol6.Pathway of Atoms in FermentationA pulse-chase experiment using 14C-labeled carbon sourcesis carried out on a yeast extract maintained under strictly anaerobic conditions to produce ethanol.The experiment consists of incubating a small amount of 14C-labeled substrate (the pulse) with theyeast extract just long enough for each intermediate in the fermentation pathway to become labeled.The label is then chased through the pathway by the addition of excess unlabeled glucose.The chaseeffectively prevents any further entry of labeled glucose into the pathway.(a)If [1-14C]glucose (glucose labeled at C-1 with 14C) is used as a substrate, what is the location of14C in the product ethanol? Explain.(b)Where would 14C have to be located in the starting glucose to ensure that all the 14C activity isliberated as 14CO2during fermentation to ethanol? Explain.AnswerAnaerobiosis requires the regeneration of NADfrom NADH in order to allow glycol-ysis to continue.(a)Figure 146 illustrates the fate of the carbon atoms of glucose.C-1 (or C-6) becomes C-3 ofglyceraldehyde 3-phosphate and subsequently pyruvate.
When pyruvate is decarboxylatedand reduced to ethanol, C-3 of pyruvate becomes the C-2 of ethanol (14CH3CH2OH).(b)If all the labeled carbon from glucose is converted to 14CO2during ethanol fermentation,the original label must have been on C-3 and/or C-4 of glucose, because these are con-verted to the carboxyl group of pyruvate.7.Heat from FermentationsLarge-scale industrial fermenters generally require constant, vigorous cool-ing.Why?AnswerFermentation releases energy, some conserved in the form of ATP but much of it dis-sipated as heat.
Unless the fermenter contents are cooled to counterbalance this heat produc-tion, the temperature would become high enough to kill the microorganisms.8.Fermentation to Produce Soy SauceSoy sauce is prepared by fermenting a salted mixture of soy-beans and wheat with several microorganisms, including yeast, over a period of 8 to 12 months.
Theresulting sauce (after solids are removed) is rich in lactate and ethanol.How are these two compoundsproduced? To prevent the soy sauce from having a strong vinegar taste (vinegar is dilute acetic acid),oxygen must be kept out of the fermentation tank.
Why?AnswerSoybeans and wheat contain starch, a polymer of glucose, which is broken down toglucose by the microorganisms.
The glucose is then degraded to pyruvate via glycolysis.
Be-cause the process is carried out in the absence of oxygen (i.e., it is a fermentation), pyruvateis reduced to lactic acid and ethanol.If oxygen were present, pyruvate would be oxidized toacetyl-CoA and then to CO2and H2O.
Some of the acetyl-CoA, however, would also be hy-drolyzed to acetic acid (vinegar) in the presence of pm
Page S-162 pinnacle OSX:Desktop 14Glycolysis, Gluconeogenesis, and the Pentose Phosphate PathwayAnswerThe transformation of glucose to lactate occurs when myocytes are low in oxygen,and it provides a means of generating ATP under oxygen-deficient conditions.Because lactatecan be transformed to pyruvate, glucose is not wasted: the pyruvate can be oxidized by aero-bic reactions when oxygen becomes plentiful.This metabolic flexibility gives the organism agreater capacity to adapt to its environment.13.Free-Energy Change for Triose Phosphate OxidationThe oxidation of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate, catalyzed by glyceraldehyde 3-phosphate dehydrogenase,proceeds with an unfavorable equilibrium constant (Keq 0.08; G 6.3 kJ/mol), yet the flowthrough this point in the glycolytic pathway proceeds smoothly.
How does the cell overcome the unfa-vorable equilibrium?AnswerIn organisms, where directional flow in a pathway is required, exergonic reactionsare coupled to endergonic reactions to overcome unfavorable free-energy changes.The ender-gonic glyceraldehyde 3-phosphate dehydrogenase reaction is followed by the phosphoglycer-ate kinase reaction, which rapidly removes the product of the former reaction.Consequently,the dehydrogenase reaction does not reach equilibrium and its unfavorable free-energychange is thus circumvented.The net Gof the two reactions, when coupled, is 18.5 kJ/mol
6.3 kJ/mol 12.2 kJ/mol.14.Arsenate PoisoningArsenate is structurally and chemically similar to inorganic phosphate (Pi), andmany enzymes that require phosphate will also use arsenate.Organic compounds of arsenate are lessstable than analogous phosphate compounds, however.For example, acyl arsenatesdecomposerapidly by the other hand, acyl phosphates,such as 1,3-bisphosphoglycerate, are more stable and undergo fur-ther enzyme-catalyzed transformation in cells.(a)Predict the effect on the net reaction catalyzed by glyceraldehyde 3-phosphate dehydrogenase ifphosphate were replaced by arsenate.(b)What would be the consequence to an organism if arsenate were substituted for phosphate? Ar-senate is very toxic to most organisms.Explain why.Answer(a)In the presence of arsenate, the product of the glyceraldehyde 3-phosphate dehydroge-nase reaction is 1-arseno-3-phosphoglycerate, which nonenzymatically decomposes to 3-phosphoglycerate and arsenate; the substrate for the phosphoglycerate kinase is there-fore bypassed.(b)No ATP can be formed in the presence of arsenate because 1,3-bisphosphoglycerate isnot formed.Under anaerobic conditions, this would result in no net glycolytic synthesisof ATP.Arsenate poisoning can be used as a test for the presence of an acyl phosphateintermediate in a reaction pathway.15.Requirement for Phosphate in Ethanol FermentationIn 1906 Harden and Young, in a series ofclassic studies on the fermentation of glucose to ethanol and CO2by extracts of brewers yeast, pm
Page S-164 pinnacle OSX:Desktop 14Glycolysis, Gluconeogenesis, and the Pentose Phosphate PathwayIn fact, the enzyme glycerol 3-phosphate dehydrogenase catalyzes this reaction (see Fig.
2117).18.Severity of Clinical Symptoms Due to Enzyme DeficiencyThe clinical symptoms of two formsof galactosemiadeficiency of galactokinase or of UDP-glucose:galactose 1-phosphate uridylyltransferaseshow radically different severity.Although both types produce gastric discomfort after milk ingestion,deficiency of the transferase also leads to liver, kidney, spleen, and brain dysfunction and eventualdeath.What products accumulate in the blood and tissues with each type of enzyme deficiency?Estimate the relative toxicities of these products from the above information.AnswerIn galactokinase deficiency, galactose accumulates; in UDP-glucose:galactose 1-phosphate uridylyltransferase deficiency, galactose 1-phosphate accumulates (see Fig.
1412).The latter metabolite is clearly more toxic.19.Muscle-Wasting in StarvationOne consequence of starvation is a reduction in muscle mass.Whathappens to the muscle proteins?AnswerMuscle proteins are selectively degraded by proteases in myocytes, and the resultingamino acids move, in the bloodstream, from muscle to liver.In the liver, glucogenic aminoacids are the starting materials for gluconeogenesis, to provide glucose for export to the brain(which cannot use fatty acids as fuel).20.Pathway of Atoms in GluconeogenesisA liver extract capable of carrying out all the normal meta-bolic reactions of the liver is briefly incubated in separate experiments with the following 14C-labeledprecursors:(a) [14C]Bicarbonate, (b) [ the pathway of each precursor through gluconeogenesis.
Indicate the location of 14C in all inter-mediates and in the product, glucose.Answer(a)In the pyruvate carboxylase reaction, 14CO2is added to pyruvate to form [4-14C]oxaloac-etate, but the phosphoenolpyruvate carboxykinase reaction removes the sameCO2inthe next step.Thus, 14C is not (initially) incorporated into pm
Page S-166 pinnacle OSX:Desktop how this reaction inhibits the transformation of lactate to pyruvate.Why does this lead tohypoglycemia?AnswerThe first step in the synthesis of glucose from lactate in the liver is oxidation of thelactate to pyruvate; like the oxidation of ethanol to acetaldehyde, this requires NAD.Con-sumption of alcohol forces a competition for NADbetween ethanol metabolism and gluco-neogenesis, reducing the conversion of lactate to glucose and resulting in hypoglycemia.
Theproblem is compounded by strenuous exercise and lack of food because at these times thelevel of blood glucose is already low.26.Blood Lactate Levels during Vigorous ExerciseThe concentrations of lactate in blood plasmabefore, during, and after a 400 m sprint are shown in the graph.Chapter 14Glycolysis, Gluconeogenesis, and the Pentose Phosphate PathwayS-169(a)What causes the rapid rise in lactate concentration?(b)What causes the decline in lactate concentration after completion of the sprint? Why does thedecline occur more slowly than the increase?(c)Why is the concentration of lactate not zero during the resting state?Answer(a)Rapid depletion of ATP during strenuous muscular exertion causes the rate of glycolysisto increase dramatically, producing higher cytosolic concentrations of pyruvate andNADH; lactate dehydrogenase converts these to lactate and NAD(lactic acidfermentation).(b)When energy demands are reduced, the oxidative capacity of the mitochondria is againadequate, and lactate is transformed to pyruvate by lactate dehydrogenase, and thepyruvate is converted to glucose
.The rate of the dehydrogenase reaction is slower inthis direction because of the limited availability of NADand because the equilibrium ofthe reaction is strongly in favor of lactate (conversion of lactate to pyruvate is energy-requiring).(c)The equilibrium of the lactate dehydrogenase reactionPyruvate NADH H88nlactate NADis stronglyin favor of lactate.Thus, even at very low concentrations of NADH and pyru-vate, there is a significant concentration of lactate.Blood [lactate] (M)0150Time ( pm
Page S-169 pinnacle OSX:Desktop of the Pentose Phosphate PathwayIf the oxidation of glucose 6-phosphate via the pentosephosphate pathway were being used primarily to generate NADPH for biosynthesis, the other product,ribose 5-phosphate, would accumulate.What problems might this cause?AnswerAt the very least, accumulation of ribose 5-phosphate would tend to force this re-action in the reverse direction by mass action (see Eqn 134, p.
493).It might also affectother metabolic reactions that involve ribose 5-phosphate as a substrate or productsuchas the pathways of nucleotide synthesis.Data Analysis Problem31.Engineering a Fermentation System Fermentation of plant matter to produce ethanol for fuel isone potential method for reducing the use of fossil fuels and thus the CO2emissions that lead to globalwarming.
Many microorganisms can break down cellulose then ferment the glucose to ethanol.How-ever, many potential cellulose sources, including agricultural residues and switchgrass, also containsubstantial amounts of arabinose, which is not as easily fermented.Chapter 14Glycolysis, Gluconeogenesis, and the Pentose Phosphate coliis capable of fermenting arabinose to ethanol, but it is not naturally tolerant ofhigh ethanol levels, thus limiting its utility for commercial ethanol production.Another bacterium,Zymomonas mobilis, is naturally tolerant of high levels of ethanol but cannot ferment arabinose.Deanda, Zhang, Eddy, and Picataggio (1996) described their efforts to combine the most useful featuresof these two organisms by introducing the E.
coligenes for the arabinose-metabolizing enzymes intoZ.mobilis.(a)Why is this a simpler strategy than the reverse: engineering E.colito be more ethanol-tolerant?Deanda and colleagues inserted five E.coligenes into the Z.mobilis genome: araA,coding for L-arabinose isomerase, which interconverts L-arabinose and L-ribulose; araB, L-ribulokinase, which usesATP to phosphorylate L-ribulose at C-5; araD, L-ribulose 5-phosphate epimerase, which interconverts L-ribulose 5-phosphate and L-xylulose 5-phosphate; talB, transaldolase; and tktA, transketolase.(b)For each of the three araenzymes, briefly describe the chemical transformation it catalyzes and,where possible, name an enzyme discussed in this chapter that carries out an analogous reaction.The fiveE.coligenes inserted in Z.
mobilisallowed the entry of arabinose into the nonoxidativephase of the pentose phosphate pathway (Fig.1422), where it was converted to glucose 6-phosphateand fermented to ethanol.(c)The three araenzymes eventually converted arabinose into which sugar?(d)The product from part (c)feeds into the pathway shown in Figure 1422.Combining the five E.colienzymes listed above with the enzymes of this pathway, describe the overall pathway forthe fermentation of 6 molecules of arabinose to ethanol.(e)What is the stoichiometry of the fermentation of 6 molecules of arabinose to ethanol and CO2?How many ATP molecules would you expect this reaction to generate?(f)Z.mobilisuses a slightly different pathway for ethanol fermentation from the one described inthis chapter.As a result, the expected ATP yield is only 1 ATP per molecule of arabinose.Although this is less beneficial for the bacterium, it is better for ethanol production. pm
Page S-171 pinnacle OSX:Desktop 14Glycolysis, Gluconeogenesis, and the Pentose Phosphate PathwayS-161The net equation isGlyceraldehyde 3-phosphate 2ADP Pi88nlactate NADG57 kJ/molBecause the payoff phase uses two glyceraldehyde 3-phosphate molecules from each glucoseentering glycolysis, the net equation is 2 Glyceraldehyde 3-phosphate 4ADP 2Pi88n2 lactate 2NADand the energetic payoff for the net reaction is G114 kJ/mol.3.GLUT TransportersCompare the localization of GLUT4 with that of GLUT2 and GLUT3, andexplainwhy these localizations are important in the response of muscle, adipose tissue, brain, andliver to insulin.AnswerGLUT2 (and GLUT1) is found in liver and is always present in the plasma membraneof hepatocytes.GLUT3 is always present in the plasma membrane of certain brain cells.GLUT4 is normally sequestered in vesicles in cells of muscle and adipose tissue and enters theplasma membrane only in response to insulin.Thus, liver and brain can take up glucose fromblood regardless of insulin level, but muscle and adipose tissue take up glucose only when in-sulin levels are elevated in response to high blood glucose.4.Ethanol Production in YeastWhen grown anaerobically on glucose, yeast (S.
cerevisiae) convertspyruvate to acetaldehyde, then reduces acetaldehyde to ethanol using electrons from NADH.Write theequation for the second reaction, and calculate its equilibrium constant at 25 C, given the standardreduction potentials in Table 137.AnswerCH3CHO NADH HCH3CH2OH NADAcetaldehydeEthanolSolve for Kequsing the Evalues in Table 137 and Equations 133 and 137.GRTlnKeqGnERTlnKeqnElnKeqIn this reaction, n2, and E0.123 V (calculated from values in Table 137 as shown inWorked Example 133).Substitute the standard values for the faraday and R, and 298 K forthe temperature:lnKeq9.58 Keqe9.58= 1.45 1045.Energetics of the Aldolase ReactionAldolase catalyzes the glycolytic reactionFructose 1,6-bisphosphate 88nglyceraldehyde 3-phosphate dihydroxyacetone phosphateThe standard free-energy change for this reaction in the direction written is 23.8 kJ/mol.The con-centrations of the three intermediates in the hepatocyte of a mammal are: fructose 1,6-bisphosphate,1.4 105M; glyceraldehyde 3-phosphate, 3 106M; and dihydroxyacetone phosphate, 1.6 105M.At body temperature (37 C), what is the actual free-energy change for the reaction?2(96,480 J/V mol)(0.123V)(8.315 J/mol K)(298 K)n pm
Page S-161 pinnacle OSX:Desktop of Triose Phosphates14C-Labeled glyceraldehyde 3-phosphate was added to a yeastextract.After a short time, fructose 1,6-bisphosphate labeled with 14C at C-3 and C-4 was isolated.What was the location of the 14C label in the starting glyceraldehyde 3-phosphate? Where did the sec-ond 14C label in fructose 1,6-bisphosphate come from? Explain.AnswerProblem 1 outlines the steps in glycolysis involving fructose 1,6-bisphosphate, glyc-eraldehyde 3-phosphate, and dihydroxyacetone phosphate.Keep in mind that the aldolase re-action is readily reversible and the triose phosphate isomerase reaction catalyzes extremelyrapid interconversion of its substrates.
Thus, the label at C-1 of glyceraldehyde 3-phosphatewould equilibrate with C-1 of dihydroxyacetone phosphate (G7.5 kJ/mol).Because thealdolase reaction has G23.8 kJ/mol in the direction of hexose formation, fructose 1,6-bisphosphate would be readily formed, and labeled in C-3 and C-4 (see Fig.146).10.Glycolysis ShortcutSuppose you discovered a mutant yeast whose glycolytic pathway was shorterbecause of the presence of a new enzyme catalyzing the reactionChapter 14Glycolysis, Gluconeogenesis, and the Pentose Phosphate PathwayS-163Would shortening the glycolytic pathway in this way benefit the cell? Explain.AnswerUnder anaerobic conditions, the phosphoglycerate kinase and pyruvate kinase reac-tions are essential.The shortcut in the mutant yeast would bypass the formation of an acylphosphate by glyceraldehyde 3-phosphate dehydrogenase and therefore would not allow theformation of 1,3-bisphosphoglycerate.Without the formation of a substrate for 3-phosphoglyc-erate kinase, no ATP would be formed.Under anaerobic conditions, the net reaction for gly-colysis normally produces 2 ATP per glucose.In the mutant yeast, net production of ATPwould be zero and growth could not occur.Under aerobic conditions, however, because themajority of ATP formation occurs via oxidative phosphorylation, the mutation would have noobservable effect.11.Role of Lactate DehydrogenaseDuring strenuous activity, the demand for ATP in muscle tissue isvastly increased.In rabbit leg muscle or turkey flight muscle, the ATP is produced almost exclusivelyby lactic acid fermentation.ATP is formed in the payoff phase of glycolysis by two reactions, promotedby phosphoglycerate kinase and pyruvate kinase.Suppose skeletal muscle were devoid of lactate dehy-drogenase.Could it carry out strenuous physical activity; that is, could it generate ATP at a high rateby glycolysis? Explain.AnswerThe key point here is that NADmust be regenerated from NADH in order for gly-colysis to continue.
Some tissues, such as skeletal muscle, obtain almost all their ATP throughthe glycolytic pathway and are capable of short-term exercise only (see Box 142).In order togenerate ATP at a high rate, the NADH formed during glycolysis must be oxidized.In the ab-sence of significant amounts of O2in the tissues, lactate dehydrogenase converts pyruvate andNADH to lactate and NAD.
In the absence of this enzyme, NADcould not be regeneratedand glycolytic production of ATP would stopand as a consequence, muscle activity couldnot be maintained.12.Efficiency of ATP Production in MuscleThe transformation of glucose to lactate in myocytes re-leases only about 7% of the free energy released when glucose is completely oxidized to CO2and H2O.Does this mean that anaerobic glycolysis in muscle is a wasteful use of glucose? Explain.Glyceraldehyde pm
Page S-163 pinnacle OSX:Desktop 14Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway27.Relationship between Fructose 1,6-Bisphosphatase and Blood Lactate LevelsA congenitaldefect in the liver enzyme fructose 1,6-bisphosphatase results in abnormally high levels of lactate inthe blood plasma.Explain.AnswerIn the liver, lactate is converted to pyruvate and then to glucose by gluconeogenesis(