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Biochemistry 102 PROBLEM SET #9 Paul Price

Gluconeogenesis/Glycogen synthesis

1. Glucogenic Substrates. A common procedure for determining the effectiveness of compounds as

precursors of glucose in mammals is to fast the animal until the liver glycogen stores are depleted andthen administer the substrate in question. A substrate that leads to a netincrease in liver glycogen is

termed glucogenic because it must first be converted to glucose-6-phosphate. Which of the following

substances are glucogenic?

(a) succinate; (b) Glycerol; (c) Acetyl CoA; (d) pyruvate; (e) CH3CH2CH2(CO) CoA; (f) Aspartate

2. Pathway of atoms in gluconeogenesis. A liver extract capable of carrying out all of the reactions of

gluconeogenesis is briefly incubated in separate experiments with the following precursors. Where

will the 14C label be located in the product glucose? (a) 14C-labeled bicarbonate (H14CO3-); (b)

pyruvate labeled with 14C in the carboxyl carbon.

3. Although palmitate cannot be used to make glucose, 14C label in palmitate will nevertheless be

incorporated into glucose. Explain this apparent paradox. Which carbon atoms of glucose will

become labeled if palmitate is labeled with 14C in carbon 16? in carbon 15?4. A congenital defect in the liver enzyme fructose 1,6-bisphosphatase results in abnormally high levels

of lactate in blood plasma. Explain.5. Blood Lactate Levels During VigorousExercise. The concentration of lactate in

blood plasma before,during, and after a 400

m sprint are shown.(a) What causes the rapid rise in lactate

concentration?

(b) What causes the decline in lactate

concentration after completion of the run?Why does the decline occur more slowly

than the increase?

(c) Why is the concentration of lactate not zeroduring the resting state?

What is the cost of storing glucose as glycogen? Write the sequence of the steps and the net reaction

required to calculate the cost in number of ATPs of converting cytoplasmic glucose 6-phosphate intoglycogen and back into glucose 6-phosphate. What fraction of the maximum number of ATPs that

are available from complete catabolism of glucose 6-phosphate does this cost represent?

7. At what point Is Glycogen Synthesis Regulated? Explain how the two following observationsidentify the point of regulation in the synthesis of glycogen in skeletal muscle.

(a) The measured activity of glycogen synthase in resting muscle, expressed in micromoles of

UDP-glucose used per gram per minute, is lower than the activity of phosphoglucomutase or UDP-glucose pyrophosphorylase, each measured in terms of micromoles of substrate transformed per gramper minute.

(b) Stimulation of glycogen synthesis leads to a small decrease in the concentrations of glucose-6-

phosphate and glucose-1-phosphate, a large decrease in the concentration of UDP-glucose, but asubstantial increase in the concentration of UDP.

8. Explain the effects of each of the following on gluconeogenesis and glycolysis in a liver cell: (a)

increasing the concentration of AMP, (b) decreasing the concentration of acetyl CoA, (c) increasingthe concentration of F2,6BP, (d) phosphorylation of PFK-2, (e) increasing the concentration of citrate,

(f) phosphorylation of fructose 2,6-bisphosphatase.

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9. The increased level of blood lactate generated by muscle cells during vigorous exercise is convertedback to glucose and glycogen in the liver. How many ATP equivalents and how many NADH

molecules must be supplied by the liver cell to convert 2 lactate molecules to glucose? to convert 2

lactate molecules to a glucose subunit of glycogen?

10. Avidin, a 70-kd protein in egg white, has a very high affinity for biotin. In fact, it is a highly specific

inhibitor of biotin enzymes. Which of the following conversions would be blocked by the addition of

avidin to a cell homogenate?

(a) Glucose pyruvate (d) Glucoseribose 5 phosphate

(b) Pyruvateglucose (e) Pyruvateoxaloacetate

(c) Oxaloacetateglucose

11. A sample of liver tissue was obtained post-mortem from the body of a patient believed to be

genetically deficient in one of the enzymes of carbohydrate metabolism. A homogenate of the liversample had the following characteristics: (1) it degraded glycogen to glucose 6-phosphate, (2) it was

unable to make glycogen from any sugar or to utilize galactose as an energy source, and (3) it

synthesized glucose 6-phosphate from lactate. Which of the following three enzymes is deficient?Give reasons for your choice.

(a) Glycogen phosphorylase (c) UDP-glucose pyrophosphorylase

(b) Fructose bisphosphatase

ANSWERS TO PROBLEM SET #9

1. (a), (b), (d), and (f) are glucogenic; (c) and (e) are not.

2. (a) In the pyruvate carboxylase reaction, 14CO2is added to pyruvate to form labeled oxaloacetate, but

phosphoenolpyruvate carboxykinase removes the same 14CO2in the next step. 14C is therefore

not incorporated into glucose.

(b) 14C label will be in carbon atoms 3 and 4 of the product glucose.

3. Fatty acids cannot be converted to glucose because the acetyl CoA units generated by FAO do not,

when they enter the TCA cycle, increase the total concentration of TCA cycle intermediates.Gluconeogenesis can only be supported by compounds, such as most amino acids, which are

catabolized to pyruvate or TCA cycle intermediates. 14C label in fatty acids does, however, enter

glucose even though fatty acids do not support net glucose synthesis. This occurs because the 14C

labeled acetate that enters citrate in the citrate synthase reaction is retained in malate. C16 labeled

palmitate yields CH3labeled acetyl CoA, and subsequently yields malate labeled in carbons 2 and 3.

These become carbons 1, 2, 5 and 6 of glucose. C15labeled palmitate yields acetyl CoA labeled in

the carboxyl carbon, and subsequently yields malate labeled in carbons 1 and 4. Carbon 4 is lost as14CO2; carbon 1 becomes carbons 3 and 4 of glucose.

4. The major pathway for the removal of lactate from blood is the transformation of lactate to glucose inthe liver by gluconeogenesis. This pathway includes the glycolytic bypass step catalyzed by fructose-

1,6-bisphphatase. A deficiency in the activity of this enzyme will therefore reduce the conversion of

lactate to glucose in hepatocytes, and thereby cause lactate to accumulate in blood plasma.

5. (a) During the 400 m sprint the demand for ATP energy to drive muscle contraction exceeds the

capacity of oxidative phosphorylation to provide energy because it is not possible to provide

sufficient O2 to the muscle cell and the cell becomes somewhat anaerobic. Under these

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circumstances the NADH/NAD+ratio in the cytosol increases substantially from its previously

low value of 0.0014. Since the equilibrium of the lactate dehydrogenase reaction:

pyruvate + NADH + H+ lactate + NAD+

strongly favors lactate, this increase in the NADH/NAD+ ratio causes a corresponding increase in

the lactate/pyruvate ratio and the concentration of blood lactate increases in the muscle cell, and

ultimately in blood. The advantage of this shift in the equilibrium ratio of lactate to pyruvate is

clear, since one NAD+is generated from each additional lactate formed. This NAD+can be used

in the 3-phosphoglycerate dehydrogenase step of glycolysis and so allows the production ofadditional ATP energy for muscle contraction.

(b) When energy demands are reduced after the sprint, the oxidative capacity of the mitochondria is

again adequate and the [NADH] / [NAD+] ratio in the muscle cell cytosol returns to its low basallevel of 0.0014, and the rate of lactate production is dramatically reduced

. The decline in blood lactate occurs slowly because it takes time for the liver cell to convert lactate back

to glucose.(c) Because some tissues (such as erythrocytes) generate all of their energy from anaerobic

glycolysis, and therefore contribute lactate to blood in the resting state. In addition, since the

equilibrium of the lactate dehydrogenase reaction strongly favors lactate, there is probably some

production of lactate by muscle cells even when the basal [NADH] / [NAD+] ratio is only 0.0014.

6. Synthesis of glycogen:

Glucose 6-phosphate glucose 1-phosphate

Glucose 1-phosphate + UTP UDP-glucose + PPi

PPi+ H2O 2Pi

UDP-glucose + glycogenn(glycogen)n+1 + UDP

UDP + ATP UTP + ADP

(1) Glucose 6-phosphate + ATP + H2O + glycogenn(glycogen)n+1 + ADP + 2Pi

Breakdown of glycogen:

Glycogenn + 1+ Pi

glucose 1-phosphate + (glycogen)nGlucose 1-phosphate _glucose-6-phosphate_______________________________

(2) Glycogenn + 1+ Pi glucose 6-phosphate+ GlycogennNet[(1) + (2)]: ATP + H2O ADP + Pi

It costs 1 mol of ATP per mole of glucose 6-phosphateglycogenglucose-6-phosphate. Foreach mole of glucose 6-phosphate completely catabolized, 31 mol of ATP are formed. The cost of

energy storage is 1/31 of this, or 3.2%, and the efficiency of storage is 96.8%.

7. The observation that glycogen synthase has the lowest measured activity of the enzymes in glycogen

synthesis suggests that this enzyme step is the rate limiting step, or bottleneck, in the flow of

metabolites, and is thus a regulatory point. This is confirmed by the observation that the stimulationof glycogen synthesis by the activation of the regulatory enzyme leads to a decrease in theconcentration of the substrate of the glycogen synthase reaction (UDP-glucose) and an increase in theconcentration of the product of the reaction (UDP).

8. (a) [AMP] allosterically inhibits gluconeogenesis at the FBPase I step ( effector is AMP), andallosterically activates glycolysis at the PFKI step (+ effector is AMP).

(b) [acetyl CoA] allosterically inhibits gluconeogenesis from pyruvate at the pyruvate carboxylasestep (+ effector is acetyl CoA), and allosterically activates glycolysis at the pyruvate kinase step

( effector is acetyl CoA). Note that [acetyl CoA] also accelerates the further catabolism ofpyruvate in two ways: by allosterically activating pyruvate dehydrogenase ( effector acetyl CoA)

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and by allosterically inhibiting pyruvate dehydrogenase kinase, which in turn shifts pyruvatedehydrogenase itself to the dephosphorylated, active state.

(c) [F2,6BP] allosterically inhibits gluconeogenesis at the FDPase I step ( effector is F2,6BP) andallosterically activates glycolysis at the PFKI step (+ effector is F2,6BP).

(d) Phosphorylation of PFKII inhibits the kinase (PFKII) and activates the phosphatase (FBPase II);

the overall effect is to reduce the F-2,6-BP concentration. Reduced F-2,6BP concentration

activates gluconeogensis and inhibits glycolysis (see (c) above).

(e) [citrate] activates gluconeogenesis at the FBPase I step (+ effector in citrate) and inhibitsglycolysis at the PFKI step ( effector is citrate).

(f) Phosphorylation of fructose-2,6-bisphosphatase (FBPase II) has the same effect as

phosphorylating PFKII, since both enzyme activities are on the same polypeptide chain and the

phosphorylation of one specific serine per chain activates FBPase II and inhibits PFKII.

9. (a) 6 ATP, no NADH. The NADH required by glyceraldehyde-3-P dehydrogenase is supplied by

lactate via lactate dehydrogenase. There are (per glucose) 2 ATP's consumed in the pyruvatecarboxylase, phosphoenol pyruvate carboxykinase, and phosphoglycerate kinase steps of

gluconeogenesis.

(b) 7 ATP, no NADH. There is an additional ATP consumed in the incorporation of glucose-6-P

into glycogen.

10. Conversions b and e would be blocked, since both require pyruvate carboxylase.

11. Identification of a Defective Enzyme in Carbohydrate Metabolism.

Characteristic (1) indicates that glycogenphosphorylase (a) and phosphoglucomutase

are functioning, while characteristic (3)

indicates that fructose bisphosphatase (b) isfunctioning. The missing enzyme is (c)

UDP-glucose pyrophosphorylase. This

enzyme is involved in the synthesis of UDP-glucose (from glucose 1-phosphate and

UTP), a key intermediate in the synthesis ofglycogen, and in the conversion of galactose

to glucose, which is necessary in order to

use galactose as an energy source.