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Labeling Studies in Isolated Mitochondria The metabolic pathways of organic compounds have often been delineated by using a radioactively labeled substrate and following the fate of the label. (a) Suppose you add a brief pulse of [3-14C] pyruvate (labeled in the methyl position) to the mitochondria. After one turn of the citric acid cycle, what is the location of the14C in the oxaloacetate?

Equally distributed in C2 and C3 of oxaloacetate, infinite number of turns

Energy Yield from the Citric Acid Cycle. The reaction catalyzed by succinyl-CoA synthetase produces the high-energy compound GTP. How is the free energy contained in GTP incorporated into the cellular ATP pool?

GTP can be transferred to ADP by a kinase. GTP + ADP --> GDP + ATP

Metabolic Effects of Mutant Enzymes Predict and explain the effect on glycogen metabolism of each of the following defects caused by mutation: defective glucagon receptors in liver

Gluconeogenesis can not be stimulated when blood glucose is low, leading to dangerously low blood glucose during periods of fasting

Why is it important that gluconeogenesis is not the exact reversal of glycolysis?

Gluconeogenesis would be highly endergonic and it would be impossible to separately regulate gluconeogenesis and glycolysis

2. Net Equation for Glycolysis and the Citric Acid Cycle Write the net biochemical equation for the metabolism of a molecule of glucose by glycolysis and the citric acid cycle, including all cofactors.

Glucose + 4 ADP + 4 Pi + 10 NAD+ + 2 FAD --> 4 ATP + 10 NADH + 2 FADH2 + 6 CO2

In what ways is glycolysis regulated by ATP levels?

Glycolytic flux is reduced when ATP is plentiful

Relationship between Energy Release and the Oxidation State of Carbon. A eukaryotic cell can use glucose (C6H12O6) and hexanoic acid (C6H14O2) as fuels for cellular respiration. On the basis of their structural formulas, which substance releases more energy per gram on complete combustion to CO2and H2O?

Hexanoic acid is more reduced and yields more energy on complete combustion to CO2 and H2O

(a) For each pathway, describe at least one control mechanism responsible for the change you predict. Tissue and Pathways 1. Adipose: fatty acid synthesis 2. Muscle: glycolysis; fatty acid synthesis; glycogen synthesis 3. Liver: glycolysis; gluconeogenesis; glycogen synthesis; fatty acid synthesis; pentose phosphate pathway

1 Adipose and 3 liver: fatty acid synthesis slower because lack of insulin results in inactive acetyl-CoA carboxylase, the first enzyme of fatty acid synthesis. Glycogen synthesis inhibited by cAMP-dependent phosphorylation of glycogen synthase. 2. Muscle: glycolysis slower because GLUT4 is inactive so glucose uptake is inhibited. 3. Liver: glycolysis slower because the bifunctional PFK2/FBPase-2 is converted to the form with active FBPase-2, decreasing F26BP

1. Citrate synthase: Acetyl-CoA + oxaloacetate + H2O --> Citrate + CoA 2. Aconitase Citrate --> Isocitrate 3. Isocitrate dehydrogenase Isocitrate + NAD+ --> a-ketoglutarate + CO2 + NADH 4. a-Ketoglutarate dehydrogenase a-Ketoglutarate + NAD+ CoA --> Succinyl-CoA + CO2 + NADH 5. Succinyl-CoA synthetase Succinyl-CoA + Pi + GDP --> Succinate + CoA + GTP 6. Succinate dehydrogenase Succinate + FAD --> Fumarate + FADH2 7. Fumarase Fumarate + H2O --> Malate 8. Malate Dehydrogenase Malate + NAD+ --> Oxaloacetate + NADH +H+

1 . Balance Sheet for the Citric Acid Cycle The citric acid cycle has eight enzymes: citrate synthase, aconitase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinyl-CoA synthetase, succinate dehydrogenase, fumarase, and malate dehydrogenase. (a) Name the cofactor(s) required by each enzyme reaction.

1. CoA, condensation 2. none, isomerization 3. NAD+, oxidative decarboxylation 4. NAD+, CoA, TPP, Oxidative decarboxylation 5. CoA, Substrate level-phosphorylation 6. FAD, oxidation 7. none, hydration 8. NAD+, oxidation

Balance Sheet for the Citric Acid Cycle The citric acid cycle has eight enzymes: citrate synthase, aconitase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinyl-CoA synthetase, succinate dehydrogenase, fumarase, and malate dehydrogenase. For each enzyme determine which of the following describes the type of reaction(s) catalyzed: condensation (carbon-carbon bond formation); dehydration (loss of water); hydration (addition of water); decarboxylation (loss of CO2); oxidation-reduction; substrate level phosphorylation; isomerization.

Respiration Studies in Isolated Mitochondria. Cellular respiration can be studied in isolated mitochondria by measuring oxygen consumption under different conditions. If 0.01 M sodium malonate is added to actively respiring mitochondria that are using pyruvate as fuel, respiration soon stops and a metabolic intermediate accumulates. (c) Explain why oxygen consumption stops.

A block in the cycle stops NADH formation, which stops electron transfer, which stops respiration

A large excess of succinate overcomes the competitive inhibition

[1-14C]Glucose Catabolism. An actively respiring bacterial culture is briefly incubated with [1-14C]glucose, and the glycolytic and citric acid cycle intermediates are isolated. · Where is the 14Cin each of the intermediates listed below? Consider only the initial incorporation of14C, in the first pass of labeled glucose through the pathways. (a) Fructose 1,6-bisphosphate___________________________________________ (b) Glyceraldehyde 3-phosphate_________________________________________ (c) Phosphoenolpyruvate_______________________________________________ (d) Acetyl-CoA_______________________________________________________ (e) Citrate__________________________________________________________ (f) α-Ketoglutarate_____________________________________________________ (g) Oxaloacetate ______________________________________________________

A. C1 B. C3 C. C3 D. C2 E. C4 F. C4 G. Equally distributed in C2 and C3

Role of the Pentose Phosphate Pathway. If the oxidation of glucose 6-phosphate via the pentose phosphate pathway were being used primarily to generate NADPH for biosynthesis, the other product, ribose 5-phosphate, would accumulate. What problems might this cause?

Accumulation of ribose 5-phosphate would reverse the direction of the reaction. It might affect other metabolic reactions that involve ribose 5-phosphate

Which ones are not glucogenic

Acetyl CoA and Butyrate

Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O --> 2 CO2 + CoA + 3 NADH + FADH2 + GTP + 2 H+

Labeling Studies in Isolated Mitochondria. The metabolic pathways of organic compounds have often been delineated by using a radioactively labeled substrate and following the fate of the label. (a) How can you determine whether glucose added to a suspension of isolated mitochondria is metabolized to CO2and H2O?

Add uniformly leveled glucose and check fo rat release of CO2

Commercial Synthesis of Citric Acid Citric acid is used as a flavoring agent in soft drinks, fruit juices, and many other foods. Worldwide, the market for citric acid is valued at hundreds of millions of dollars per year. Commercial production uses the mold Aspergillus niger, which metabolizes sucrose under carefully controlled conditions. (a) Does the commercial process require the culture medium to be aerated—that is, is this a fermentation or an aerobic process? Explain.

Aerobic and requires molecules oxygen. The overall reaction consumes NAD+. NADH must be regenerated.

Commercial Synthesis of Citric Acid. Citric acid is used as a flavoring agent in soft drinks, fruit juices, and many other foods. Worldwide, the market for citric acid is valued at hundreds of millions of dollars per year. Commercial production uses the mold Aspergillus niger, which metabolizes sucrose under carefully controlled conditions. (a) The yield of citric acid is strongly dependent on the concentration of FeCl3 in the culture medium, as indicated in the graph. Why does the yield decrease when the concentration of Fe3+is above or below the optimal value of 0.5 mg/L?

Any concentration above or below the value will restrict the synthesis of aconitase

Why did the substitution of arsenate for phosphate prevent the accumulation of the hexose bisphosphate yet allow fermentation to ethanol and CO2 to go to completion? (See Problem 14.)

Arsenate replaces Pi in the glyceraldehyde 3-phosphate dehydrogenase reaction to yield an acyl arsenate which spontaneously hydrolyzes. This prevents formation of ATP, but 3-phosphoglycerate continues through the pathway.

Oxaloacetate Pool. What factors might decrease the pool of oxaloacetate available for the activity of the citric acid cycle? How can the pool of oxaloacetate be replenished?

Aspartate synthesis or gluconeogensis can decrease the pool. Oxaloacetate can be replenished by PEP carboxykinase, PEP carboxylase, magic enzyme, or pyruvate carboxylase

To prevent the soy sauce from having a strong vinegary taste (vinegar is dilute acetic acid), oxygen must be kept out of the fermentation tank. Why?

Because the process is carried out in the absence of O2, pyruvate converts to lactic acid and to ethanol. If O2 were present, pyruvate would be oxidized to acetyl CoA and then to CO2 and H2O.

Glycogen Breakdown in Migrating Birds Unlike the rabbit with its short dash, migratory birds require energy for extended periods of time. For example, ducks generally fly several thousand miles during their annual migration. The flight muscles of migratory birds have a high oxidative capacity and obtain the necessary ATP through the oxidation of acetyl-CoA (obtained from fats) via the citric acid cycle. Compare the regulation of muscle glycolysis during short-term intense activity, as in the fleeing rabbit, and during extended activity, as in the migrating duck. Why must the regulation in these two settings be different?

The migrating bird relies on the highly efficient aerobic oxidation of fats, rather than the anaerobic metabolism of glucose used by a sprinting rabbit. The bird reserves its muscle glycogen for short bursts of energy during emergencies.

Cellular Glucose Concentration The concentration of glucose in human blood plasma is maintained at about 5 mM. The concentration of free glucose inside a myocyte is much lower. Why is the concentration so low in the cell? What happens to glucose after entry into the cell? Glucose is administered intravenously as a food source in certain clinical situations. Given that the transformation of glucose to glucose 6-phosphate consumes ATP, why not administer intravenous glucose 6-phosphate instead?

The phosphate group of glucose 6-phosphate is completely ionized at pH 7, giving the molecule an overall negative charge. Because membranes are impermeable to electrically charged molecules, glucose 6-phosphate cannot pass from the bloodstream into cells and hence cannot enter the glycolytic pathway and generate ATP

One consequence of starvation is a reduction in muscle mass. What happens to the muscle proteins?

The proteins are degraded to amino acids and are used for gluconeogenesis

What causes the rapid rise in lactate concentration

The rise of pyruvate and NADH results in a rise in lactate

3 . Effect of O2 Supply on Glycolytic Rates. The regulated steps of glycolysis in intact cells can be identified by studying the catabolism of glucose in whole tissues or organs. For example, the glucose consumption by heart muscle can be measured by artificially circulating blood through an isolated intact heart and measuring the concentration of glucose before and after the blood passes through the heart. If the circulating blood is deoxygenated, heart muscle consumes glucose at a steady rate. When oxygen is added to the blood, the rate of glucose consumption drops dramatically, then is maintained at the new, lower rate. Explain.

In the absence of O2, the ATP needs are met by anaerobic glucose metabolism. Because aerobic oxidation of glucose produces more ATP than fermentation, less glucose is needed to produce the same amount of ATP

Why does the Vmax of the muscle enzyme need to be greater than that of the liver enzyme?

In working muscle, ATP flux requirements are very high and glucose 1-phosphate must be produced rapidly requiring a high Vmax

Effect of [NADH]/[NAD+] on the Citric Acid Cycle How would you expect the operation of the citric acid cycle to respond to a rapid increase in the [NADH]/[NAD+] ratio in the mitochondrial matrix? Why?

Increased NADH/NAD+ inhibits the citric acid cycle by the mass action at three NAD+ reducing steps. High NADH shifts equilibrium toward NAD+

Why does the decline occur more slowly than the increase?

It's a slower process because formation of pyruvate is limited by NAD+ availability. The lactate dehydrogenase equilibrium is in favor of lactate.

Thiamine Deficiency. Individuals with a thiamine-deficient diet have relatively high levels of pyruvate in their blood. Explain this in biochemical terms.

Lack of TPP, inhibits pyruvate dehydrogenase, pyruvate accumulates

Relationship between Fructose 1,6-Bisphosphatase and Blood Lactate Levels. A congenital defect in the liver enzyme fructose 1,6-bisphosphatase results in abnormally high levels of lactate in the blood plasma. Explain.

Lactate is transformed to glucose in the liver by gluconeogenesis. A defect in FBPase-1 would prevent entry of lactate into the gluconeogenic pathway in hepatocytes, causing lactate to accumulate in the blood

What causes the decline in lactate concentration after completion of the sprint?

Lactate is transformed to glucose via pyruvate.

Respiration Studies in Isolated Mitochondria Cellular respiration can be studied in isolated mitochondria by measuring oxygen consumption under different conditions. If 0.01 M sodium malonate is added to actively respiring mitochondria that are using pyruvate as fuel, respiration soon stops and a metabolic intermediate accumulates. (b) Explain why it accumulates.

Malonate is inhibitor of succinate dehydrogenase.

Pyruvate Dehydrogenase Cofactors and Mechanism. Describe the role of each cofactor involved in the reaction catalyzed by the pyruvate dehydrogenase complex.

NAD+ oxidizes FAD FAD: Oxidizes lipoic acid Lipoic acid: Oxidizes pyruvate to acetyl-CoA and activates a thioester CoA-SH: Activates acetate as thioester TPP: Adds an alpha carbon of pyruvate then stabilizes the carbanion

Equation for the Preparatory Phase of Glycolysis

Net Equation: Glucose + 2 ATP 2 Glyceraldehyde 3-phosphate + 2 ADP + 2H+ Delta G = 2.1 kJ/mol

Role of the Vitamin Niacin Adults engaged in strenuous physical activity require an intake of about 160 g of carbohydrate daily but only about 20 mg of niacin for optimal nutrition. Given the role of niacin in glycolysis, how do you explain the observation?

Niacin is used to synthesize NAD+. One molecule of NAD+ can oxidize many thousands of molecules of glucose. Thus, the amount required of NAD+ is relatively small.

Suppose skeletal muscle were devoid of lactate dehydrogenase. Could it carry out strenuous physical activity; that is, could it generate ATP at a high rate by glycolysis? Explain.

No because lactate dehydrogenase is required to recycle NAD+ from the NADH formed during oxidation of glyceraldehyde 3-phosphate

Would shortening the glycolytic pathway in this way benefit the cell? Explain.

No, because there would be no anaerobic production of ATP. Aerobic ATP production would be diminished only slightly

Synthesis of Oxaloacetate by the Citric Acid Cycle. Oxaloacetate is formed in the last step of the citric acid cycle by the NAD+-dependent oxidation of L-malate. Can a net synthesis of oxaloacetate from acetyl-CoA occur using only the enzymes and cofactors of the citric acid cycle, without depleting the intermediates of the cycle? Explain. How is oxaloacetate that is lost from the cycle (to biosynthetic reactions) replenished?

No. For every two carbons that enter as acetate, two leave the cycle as CO2. No net synthesis of oxaloacetate. Net synthesis of oxaloacetate occurs by the carboxylation of pyruvate

Reactions of the Pyruvate Dehydrogenase Complex. Two of the steps in the oxidative decarboxylation of pyruvate (steps 4 and 5 in Fig. 16-6) do not involve any of the three carbons of pyruvate yet are essential to the operation of the PDH complex. Explain.

Steps 4 and 5 are essential in the deoxidation of the enzyme's reduced lipoamide cofactor

Succinate

How is the succinate transformed to glucose? Explain the stoichiometry.

Succinate is transformed oxaloacetate. This passes into the cytosol and is converted to PEP by PEP carboxykinase. Two moles of PEP are required to produce a mole of glucose

Which ones are glucogenic

Succinate, glycerol, pyruvate

Regulation of Citrate Synthase. In the presence of saturating amounts of oxaloacetate, the activity of citrate synthase from pig heart tissue shows a sigmoid dependence on the concentration of acetyl-CoA, as shown in the graph below. When succinyl-CoA is added, the curve shifts to the right and the sigmoid dependence is more pronounced. On the basis of these observations, suggest how succinyl-CoA regulates the activity of citrate synthase (Hint: see fig 6-35). · Why is succinyl-CoA an appropriate signal for regulation of the citric acid cycle?

Succinyl CoA is an intermediate of the citric acid cycle. It accumulation signals reduced flux through the cycle, calling for reduced entry of acetyl CoA into the cycle

The inhibition of PFK-1 by ATP is diminished when the ADP concentration is high, as shown in the graph. How can this observation be explained?

The graph indicates that increases ADP suppresses the inhibition by ATP. Consumption of ATP leads to an increase in ADP.

(a) Explain how ATP can be both a substrate and an inhibitor of PFK-1. How is the enzyme regulated by ATP?

There are two binding sites for ATP: a catalytic site and a regulatory site. Binding of ATP to a regulatory site inhibits PFK-1, by reducing V max or increasing Km for ATP at the catalytic site.

Role of the Vitamin. Thiamine People with beriberi, a disease caused by thiamine deficiency, have elevated levels of blood pyruvate and α-ketoglutarate, especially after consuming a meal rich in glucose. How are these effects related to a deficiency of thiamine?

Thiamine is required for the synthesis of TPP. A deficiency reduces the activity of the enzyme complexes and causes the observed accumulation of precursors

which galactosemia deficiency is more toxic

UDP-Glucose deficiency is more while galactokinase deficiency is less toxic

Was the conversion of pyruvate to ethanol and CO2 essential? Why?

Yes because pyruvate must be converted to ethanol to produce a continuous supply of NAD+ for the oxidation of glyceraldehyde 3-phosphate

Oxaloacetate Depletion. Mammalian liver can carry out gluconeogenesis using oxaloacetate as the starting material (Chapter 14). Would the operation of the citric acid cycle be affected by extensive use of oxaloacetate for gluconeogenesis? Explain your answer.

Yes. The citric acid cycle would be inhibited. Removing oxaloacetate for gluconeogenesis would shift the citrate synthase reaction toward oxaloacetate

Mode of Action of the Rodenticide Fluoroacetate. Fluoroacetate, prepared commercially for rodent control, is also produced by a South African plant. After entering a cell, fluoroacetate is converted to fluoroacetyl-CoA in a reaction catalyzed by the enzyme acetate thiokinase: a. Where did the block in the citric acid cycle occur? What caused citrate to accumulate and the other cycle intermediates to be depleted? b. What is the structure of the end product of fluoroacetate metabolism? Why does it block the citric acid cycle. How might the inhibition overcome? c. Why did glucose uptake and glycolysis decrease in the heart perfusion experiment? Why did hexose monophosphate accumulate? d. Why is fluoroacetate poisoning fatal?

a. Inhibition of aconitase b. Fluorocitrate, competes with citrate, by a large excess of citrate c. Citrate and fluorocitrate are inhibitors of PFK-1 d. All catabolic processes necessary for ATP production are shut down

Blood Metabolites in Insulin Insufficiency For the patient described in Problem 12, predict the levels of the following metabolites in his blood before treatment in the emergency room, relative to levels maintained during adequate insulin treatment: (a) free fatty acids

elevated

Why did fermentation cease when the supply of phosphate was exhausted

ethanol fermentation requires 2 moles of Pi per mole of glucose

Why did ethanol and CO2 accumulate

ethanol is the reduced product formed during deoxidation of NADH to NAD+. CO2 is the byproduct of the conversion of pyruvate to ethanol

What is the cost (in ATP equivalents) of transforming glucose to pyruvate via glycolysis and back again to glucose via gluconeogenesis?

4 ATP per glucose molecule

What would be the consequence to an organism if arsenate were substituted for phosphate? Arsenate is very toxic to most organisms. Explain why.

there would be no net ATP synthesis under anaerobic conditions if arsenate was present

If [1-14C] glucose (glucose labeled at C-1 with14C) is used as a substrate, what is the location of14C in the product ethanol?

14 CH3CH2OH

(a) For each tissue listed below, is each pathway faster, slower, or unchanged in this patient, compared with the normal level when he is getting appropriate amounts of insulin? Tissue and Pathways 1. Adipose: fatty acid synthesis 2. Muscle: glycolysis; fatty acid synthesis; glycogen synthesis 3. Liver: glycolysis; gluconeogenesis; glycogen synthesis; fatty acid synthesis; pentose phosphate pathway

1: Adipose: fatty acid synthesis slower. 2. Muscle glycolysis, fatty acid synthesis, and glycogen synthesis slower. 3. Liver: glycolysis faster, gluconeogenesis, glycogen synthesis, and fatty acid synthesis slow. Pentose phosphate pathway unchanged

The payoff phase of glycolysis in skeletal muscle phosphate is converted to pyruvate (the payoff phase of glycolysis) and the pyruvate is reduced to lactate. Write balanced biochemical equations for all the reactions in this process

2 Glyceraldehyde 3-phosphate + 4 ADP + 2Pi 2 lactate + 2 NAD+

(a) Predict the effect on the net reaction catalyzed by glyceraldehyde 3-phosphate dehydrogenase if phosphate were replaced by arsenate.

3-phosphoglycerate is the product

Where would 14C have to be located in the starting glucose to ensure that all the 14C activity is liberated as 14CO2 during fermentation to ethanol? Explain.

3rd or 4th carbon

Enzyme Defects in Carbohydrate Metabolism Summaries of four clinical case studies follow. For each case determine which enzyme is defective and designate the appropriate treatment, from the lists provided at the end of the problem. Justify your choices. Answer the questions contained in each case study. (You may need to refer to information in Chapter 14.) Case AThe patient develops vomiting and diarrhea shortly after milk ingestion. A lactose tolerance test is administered. (The patient ingests a standard amount of lactose, and the glucose and galactose concentrations in blood plasma are measured at intervals. In individuals with normal carbohydrate metabolism, the levels increase to a maximum in about 1 hour, then decline.) The patient's blood glucose and galactose concentrations do not increase during the test. · Why do blood glucose and galactose increase and then decrease during the test in healthy individuals? · Why do they fail to rise in the patient? Defective Enzyme (a) Muscle PFK-1 (b) Phosphomannose isomerase (c) Galactose 1-phosphate uridylyltransferase (d) Liver glycogen phosphorylase (e) Triose kinase (f) Lactase in intestinal mucosa (g) Maltase in intestinal mucosa (h) Muscle debranching enzyme Treatment 1. Jogging 5 km each day 2. Fat-free diet 3. Low-lactose diet 4. Avoiding strenuous exercise 5. Large doses of niacin (the precursor of NAD+) 6. Frequent feedings (smaller portions) of a normal diet

A: f, 3

Cofactors for the Citric Acid Cycle. Suppose you have prepared a mitochondrial extract that contains all the soluble enzymes of the matrix but has lost (by dialysis) all the low molecular weight cofactors. What must you add to the extract so that the preparation will oxidize acetyl-CoA to CO2?

ADP, CoA-SH, TPP, NAD+, Pi

Enzyme Defects in Carbohydrate Metabolism Summaries of four clinical case studies follow. For each case determine which enzyme is defective and designate the appropriate treatment, from the lists provided at the end of the problem. Justify your choices. Answer the questions contained in each case study. (You may need to refer to information in Chapter 14.) The patient develops vomiting and diarrhea after ingestion of milk. His blood is found to have a low concentration of glucose but a much higher than normal concentration of reducing sugars. The urine tests positive for galactose. · Why is the concentration of reducing sugar in the blood high? · Why does galactose appear in the urine? Defective Enzyme (a) Muscle PFK-1 (b) Phosphomannose isomerase (c) Galactose 1-phosphate uridylyltransferase (d) Liver glycogen phosphorylase (e) Triose kinase (f) Lactase in intestinal mucosa (g) Maltase in intestinal mucosa (h) Muscle debranching enzyme Treatment 1. Jogging 5 km each day 2. Fat-free diet 3. Low-lactose diet 4. Avoiding strenuous exercise 5. Large doses of niacin (the precursor of NAD+) 6. Frequent feedings (smaller portions) of a normal diet

B: c, 3

Efficiency of ATP Production in Muscle The transformation of glucose to lactate in myocytes releases only about 7% of the free energy released when glucose is completely oxidized to CO2 and H2O. Does this mean that anaerobic glycolysis in muscle is a wasteful use of glucose? Explain.

Because lactate can be oxidized to pyruvate, glucose is not wasted. Pyruvate is oxidized by aerobic reactions when O2 becomes plentiful

What was the location of the 14C label in the starting glyceraldehyde 3-phosphate? Where did the second 14C label in fructose 1,6-bisphosphate come from? Explain.

C1 because C1 of glyceraldehyde 3-phosphate is equivalent to C4 of fructose 1,6-bisphosphate. C3 of dihydroxyacetone phosphate becomes labeled through the triode phosphate isomerase reaction.

Enzyme Defects in Carbohydrate Metabolism Summaries of four clinical case studies follow. For each case determine which enzyme is defective and designate the appropriate treatment, from the lists provided at the end of the problem. Justify your choices. Answer the questions contained in each case study. (You may need to refer to information in Chapter 14.) The patient complains of painful muscle cramps when performing strenuous physical exercise but has no other symptoms. A muscle biopsy indicates a muscle glycogen concentration much higher than normal. · Why does glycogen accumulate? Defective Enzyme (a) Muscle PFK-1 (b) Phosphomannose isomerase (c) Galactose 1-phosphate uridylyltransferase (d) Liver glycogen phosphorylase (e) Triose kinase (f) Lactase in intestinal mucosa (g) Maltase in intestinal mucosa (h) Muscle debranching enzyme Treatment 1. Jogging 5 km each day 2. Fat-free diet 3. Low-lactose diet 4. Avoiding strenuous exercise 5. Large doses of niacin (the precursor of NAD+) 6. Frequent feedings (smaller portions) of a normal diet

C: h, 4

How does the regulation of citrate synthase control the rate of cellular respiration in pig heart tissue?

Citrate synthase regulates the supply of NADH and the flow of electrons from NADH to O2

The first step in the metabolism of ethanol by the liver is oxidation to acetaldehyde, catalyzed by liver alcohol dehydrogenase: Explain how this reaction inhibits the transformation of lactate to pyruvate. Why does this lead to hypoglycemia?

Consumption of alcohol forces competition for NAD+ between ethanol metabolism and gluconeogenesis.

Enzyme Defects in Carbohydrate Metabolism Summaries of four clinical case studies follow. For each case determine which enzyme is defective and designate the appropriate treatment, from the lists provided at the end of the problem. Justify your choices. Answer the questions contained in each case study. (You may need to refer to information in Chapter 14.) The patient is lethargic, her liver is enlarged, and a biopsy of the liver shows large amounts of excess glycogen. She also has a lower than normal blood glucose level. · What is the reason for the low blood glucose in this patient? Defective Enzyme (a) Muscle PFK-1 (b) Phosphomannose isomerase (c) Galactose 1-phosphate uridylyltransferase (d) Liver glycogen phosphorylase (e) Triose kinase (f) Lactase in intestinal mucosa (g) Maltase in intestinal mucosa (h) Muscle debranching enzyme Treatment 1. Jogging 5 km each day 2. Fat-free diet 3. Low-lactose diet 4. Avoiding strenuous exercise 5. Large doses of niacin (the precursor of NAD+) 6. Frequent feedings (smaller portions) of a normal diet

D: d, 6

Synthesis of Glycerol Phosphate. The glycerol 3-phosphate required for the synthesis of glycerophospholipids can be synthesized from a glycolytic intermediate. Propose a reaction sequence for this conversion.

Dihydroxyacetone phosphate + NADH + H+ --> Glycerol 3-phosphate + NAD+. Catalyzed by dehydrogenase

Riboflavin Deficiency. How would a riboflavin deficiency affect the functioning of the citric acid cycle? Explain your answer.

FAD is required in the citric acid cycle. A deficiency would inhibit the cycle

Heat from Fermentations Large-scale industrial fermenters generally require constant, vigorous cooling. Why?

Fermentation releases energy, some conserved in the form of ATP but much of it dissipated as heat. Unless the fermenter contents are cooled, the temperature would become high enough to kill the microorganisms.

Identify the hexose bisphosphate that accumulated. Why did it accumulate?

Fructose 1,6-bisphosphate because it is formed as an intermediate in glycolysis.

Excess O2 Uptake during Gluconeogenesis Lactate absorbed by the liver is converted to glucose, with the input of 6 mol of ATP for every mole of glucose produced. The extent of this process in a rat liver preparation can be monitored by administering [14C] lactate and measuring the amount of [14C] glucose produced. Because the stoichiometry of O2 consumption and ATP production is known (about 5 ATP per O2), we can predict the extra O2consumption above the normal rate when a given amount of lactate is administered. However, when the extra O2 used in the synthesis of glucose from lactate is actually measured, it is always higher than predicted by known stoichiometric relationships. Suggest a possible explanation for this observation.

If catabolic and anabolic pathways of glucose metabolism occur simultaneously, unproductive cycling of ADP and ATP would occur. Leads to extra O2 consumption

What is the physiological function of glycogen phosphorylase in skeletal muscle? In liver tissue?

In muscle: Glycogen breakdown supplies energy via glycolysis. Glycogen phosphorylase catalyzes the conversion of stored glycogen to glucose 1-phosphate, which is converted to glucose 6-phosphate, an intermediate in glycolysis. During vigorous activity, skeletal muscle requires large quantities of glucose 6-phosphate In the liver: Glycogen breakdown maintains a steady level of blood glucose between meals

Isocitrate Dehydrogenase Reaction. What type of chemical reaction is involved in the conversion of isocitrate to α-ketoglutarate? Name and describe the role of any cofactors. What other reaction(s) of the citric acid cycle are of this same type?

Oxidative decarboxylation, NAD+ or NADP+, a-ketoglutarate dehydrogenase reaction

Relationship between Respiration and the Citric Acid Cycle. Although oxygen does not participate directly in the citric acid cycle, the cycle operates only when O2is present. Why?

Oxygen is needed to recycle NAD+ from the NADH produced by the citric acid cycle. Deoxidation of NADH occurs during mitochondrial oxidative phosphorylation

Q is much lower than K

PFK-1 is far from equilibrium. This reaction is slow and flux is determined by PFK-1

Metabolic Effects of Mutant Enzymes Predict and explain the effect on glycogen metabolism of each of the following defects caused by mutation: (a) loss of the cAMP-binding site on the regulatory subunit of protein kinase A (PKA);

PKA cannot be activated in response to glucagon or epinephrine. Glycogen phosphorylase is not activated.

Metabolic Effects of Mutant Enzymes Predict and explain the effect on glycogen metabolism of each of the following defects caused by mutation: loss of the protein phosphatase inhibitor (inhibitor 1 in Fig. 15-42);

PP1 remains active, allowing it to dephosphorylate glycogen synthase (activating it) and glycogen phosphorylase (Inhibiting it)

Metabolic Effects of Mutant Enzymes Predict and explain the effect on glycogen metabolism of each of the following defects caused by mutation: overexpression of phosphorylase b kinase in liver

Phosphorylase remains phosphorylated (active,) increasing the breakdown of glycogen

Glycogen Breakdown in Rabbit Muscle. The intracellular use of glucose and glycogen is tightly regulated at four points. To compare the regulation of glycolysis when oxygen is plentiful and when it is depleted, consider the utilization of glucose and glycogen by rabbit leg muscle in two physiological settings: a resting rabbit, with low ATP demands, and a rabbit that sights its mortal enemy, the coyote, and dashes into its burrow. For each setting, determine the relative levels (high, intermediate, or low) of AMP, ATP, citrate, and acetyl-CoA and describe how these levels affect the flow of metabolites through glycolysis by regulating specific enzymes. In periods of stress, rabbit leg muscle produces much of its ATP by anaerobic glycolysis (lactate fermentation) and very little by oxidation of acetylCoA derived from fat breakdown.

Resting: ATP is high, AMP is low, acetyl coA and citrate intermediate. Running: ATP is intermediate, AMP high, acetyl CoA and citrate low. Glucose flux through glycolysis increases during the anaerobic sprint because the ATP inhibition of glycogen phosphorylase and PFK-1 is partially relieved, AMP stimulus both enzymes, and citrate and acetyl CoA levels relieve their inhibitory effects on PFK-1 and pyruvate kinase

Fermentation to Produce Soy Sauce is prepared by fermenting a salted mixture of soybeans and wheat with several microorganisms, including yeast, over a period of 8 to 12 months. The resulting sauce (after solids are removed) is rich in lactate and ethanol. How are these two compounds produced?

Soybeans and wheat contain starch, a polymer of glucose. The microorganisms break down starch to glucose and then glucose to pyruvate.

Stimulation of Oxygen Consumption by Oxaloacetate and Malate. In the early 1930s, Albert Szent-Györgyi reported the interesting observation that the addition of small amounts of oxaloacetate or malate to suspensions of minced pigeon breast muscle stimulated the oxygen consumption of the preparation. Surprisingly, the amount of oxygen consumed was about seven times more than the amount necessary for complete oxidation (to CO2and H2O) of the added oxaloacetate or malate. Why did the addition of oxaloacetate or malate stimulate oxygen consumption? Why was the amount of oxygen consumed so much greater than the amount necessary to completely oxidize the added oxaloacetate or malate?

The addition of oxaloacetate or malate stimulates the Citric Acid Cycle and stimulates respiration. It serves as a catalytic role.

The oxidation of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate, catalyzed by glyceraldehyde 3-phosphate dehydrogenase, proceeds with an unfavorable equilibrium constant ( = 0.08; ΔG′° = 6.3 kJ/mol), yet the flow through this point in the glycolytic pathway proceeds smoothly. How does the cell overcome the unfavorable equilibrium?

The cell rapidly removes the 1,3-bisphosphoglycerate in a favorable step due to phosphoglycerate kinase enzyme

Explain in bioenergetic terms how the conversion of pyruvate to phosphoenolpyruvate in gluconeogenesis overcomes the large, negative, standard free-energy change of the pyruvate kinase reaction in glycolysis.

The cell spends 1 ATP and 1 GTP in converting pyruvate to PEP

Citric Acid Cycle Mutants. There are many cases of human disease in which one or another enzyme activity is lacking due to genetic mutation. However, cases in which individuals lack one of the enzymes of the citric acid cycle are extremely rare. Why?

The citric acid cycle is so central to metabolism that a serious defect in any cycle enzyme would probably be lethal to the embryo

Hormonal Control of Metabolic Fuel Between your evening meal and breakfast, your blood glucose drops and your liver becomes a net producer rather than consumer of glucose. Describe the hormonal basis for this switch, and explain how the hormonal change triggers glucose production by the liver

The drop in blood glucose triggers release of glucagon by the pancreas. In the liver, glucagon activates glycogen phosphorylase by stimulating its cAMP-dependent phosphorylation and stimulates gluconeogenesis by lowering F26BP, which stimulates FBPase-1

Why is the concentration of lactate not zero during the resting state?

The equilibrium for the lactate dehydrogenase reaction is in favor of lactate formation

Altered Metabolism in Genetically Manipulated Mice Researchers can manipulate the genes of a mouse so that a single gene in a single tissue either produces an inactive protein (a "knockout" mouse) or produces a protein that is always (constitutively) active. What effects on metabolism would you predict for mice with the following genetic changes: (a) knockout of glycogen debranching enzyme in the liver; (b) knockout of hexokinase IV in liver; (c) knockout of FBPase-2 in liver; (d) constitutively active FBPase-2 in liver; (e) constitutively active AMPK in muscle; (f) constitutively active ChREBP in liver?

a. Reduced capacity to mobilize glycogen, lowered blood glucose between meals B. Reduced capacity to lower blood glucose after a carb meal, elevated blood glucose C. Reduced F26BP in liver, stimulates glycolysis and inhibits gluconeogenesis D. Reduced F26BP, stimulating gluconeogenesis and inhibiting glycolysis E: Increased uptake of fatty acids and glucose, increasing oxidation of both F: Increased conversion of pyruvate to acetyl-CoA, increased fatty acid synthesis

Determine whether oxidation or reduction has occurred a. Methanol --> formaldehyde b. Formaldehyde --> Formate c. CO2 + H2 --> Formate d. Glycerinate --> Glyceraldehyde e. Glycerol --> dihydroxyacetone f. 2 H2O + Toluene --> Benzoate g. Succinate --> Fumarate h. Pyruvate + H2O --> Acetate

a. oxidation b. oxidation c. reduction d. reduction e. oxidation f. oxidation g. oxidation h. oxidation

Nicotinamide Coenzymes as Reversible Redox Carriers. The nicotinamide coenzymes (see Fig. 13-24) can undergo reversible oxidation-reduction reactions with specific substrates in the presence of the appropriate dehydrogenase. In these reactions, NADH + H+ serves as the hydrogen source, as described in Problem 3. Whenever the coenzyme is oxidized, a substrate must be simultaneously reduced: · For each of the reactions in (a) through (f) shown below, determine whether the substrate has been oxidized or reduced or is unchanged in oxidation state(see Problem 3). If a redox change has occurred, balance the reaction with the necessary amount of NAD+, NADH, H+, and H2O. · The objective is to recognize when a redox coenzyme is necessaryin a metabolic reaction. A. Ethanol + NAD+ --> Acetaldehyde + NADH B. 1,3-BPG + NADH --> Glyceraldehyde 3-P + NAD+ + HPO42- C. Pyruvate --> Acetaldehyde + CO2 D. Pyruvate + NAD+ --> Acetate + CO2 + NADH E. Oxaloacetate + NADH --> Malate + NAD+ F. Acetoacetate --> Acetone + CO2

a. oxidized b. reduced c. unchanged d. oxidized e. reduced f. unchanged

Labeling Studies in Isolated Mitochondria The metabolic pathways of organic compounds have often been delineated by using a radioactively labeled substrate and following the fate of the label. (a) Explain by tracing the14C label through the pathway. How many turns of the cycle are required to release all the [3-14C]pyruvate as CO2?

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