LEHNINGER PRINCIPLES OF BIOCHEMISTRY - VOL 2. BIOENERGETICS AND METABOLISM - 2014

PART II. BIOENERGETICS AND METABOLISM

15. PRINCIPLES OF METABOLIC REGULATION

Questions and Problems

1. Measurement of intracellular metabolite concentrations.

Measuring the concentrations of metabolic intermediates in living Cells presents significant experimental challenges; typically, Cells must be disrupted to determine a metabolite's concentration. Because enzymatic conversions of metabolites occur extremely rapidly, measurements often yield the equilibrium rather than the physiological concentration of a metabolite. Therefore, reliable results require an experimental technique that instantly arrests all enzymatic reactions in intact tissue, thereby preventing further transformations of metabolic intermediates. This is achieved by rapidly placing the tissue between large aluminum plates cooled in liquid nitrogen (-190 °C); this method is known as freeze-clamping. Following freezing, which instantly halts enzymatic activity, the tissue is pulverized, and Enzymes are inactivated by precipitation with perchloric acid. The precipitate is removed by centrifugation, and the metabolite content is determined in the clear supernatant. To calculate intracellular metabolite concentrations, the volume of the intracellular space is determined based on the total Water content of the tissue and the extracellular volume.

The table lists the intracellular concentrations of the substrates and products of the reaction catalyzed by Phosphofructokinase-1 in rat Heart tissue.

* Units are µmol/mL of intracellular water.

a) Calculate the mass-action ratio for the reaction catalyzed by PFK-1 under physiological conditions.

б) ∆G′ = -14.2 kJ/mol. Calculate the Equilibrium Constant for the reaction catalyzed by PFK-1.

c) Compare The values of Q and K′eq. Is this physiological reaction close to equilibrium? Explain your answer. What does this experiment reveal about The Role of PFK-1 as a regulatory enzyme?

2. Are metabolic reactions at equilibrium?

a) Phosphoenolpyruvate (PEP) is one of two phosphoryl group Donors for ATP synthesis during Glycolysis. In human erythrocytes, the steady-state concentration of ATP is 2.24 mM, ADP is 0.25 mM, and Pyruvate is 0.051 mM. Determine the concentration of PEP at 25 °C, assuming that the reaction catalyzed by pyruvate kinase (see Fig. 13-13) occurs under equilibrium conditions within The Cell.

b) The physiological concentration of PEP in human erythrocytes is 0.023 mM. Compare this value with your answer to part (a). How can you account for the difference between these values?

3. Effect of oxygen availability on The rate of glycolytic reactions.

Regulated steps of glycolysis in intact cells can be identified by studying Glucose Catabolism in whole Tissues and Organs. For example, glucose consumption by cardiac Muscle can be determined by artificially perfusing an isolated heart with Blood and measuring the glucose concentration in the blood entering and leaving The Heart. When anoxic blood is perfused through the heart, the muscle consumes glucose at a steady rate. When the blood is oxygenated, the rate of glucose consumption drops sharply and then stabilizes at a new, lower level. Explain this phenomenon.

4. Regulation of phosphofructokinase-1.

The Effect of ATP on The activity of the allosteric enzyme PFK-1 is shown in the figure. At a given concentration of fructose 6-phosphate, PFK-1 activity increases with rising ATP concentration; however, beyond a certain point, further increases in ATP inhibit the enzyme's activity.

a) Explain why ATP acts as both a substrate and an inhibitor of PFK-1. How does ATP exert its regulatory effect on the enzyme?

b) How is glycolysis regulated in response to cellular ATP levels?

c) The inhibition of PFK-1 by ATP is relieved at high concentrations of ADP, as shown in the figure. How would you explain this phenomenon?

5. Intracellular glucose concentration.

The concentration of glucose in human Blood Plasma is maintained at around 5 mM, whereas the concentration of free glucose in myocytes is significantly lower. Why is the intracellular glucose concentration low? What happens to glucose once it enters the cell? Some patients are prescribed intravenous glucose infusions as a source of Nutrition. The conversion of glucose to glucose-6-phosphate requires ATP consumption, so why are patients not given intravenous glucose-6-phosphate instead of glucose?

6. Enzymatic activity and physiological function.

For Glycogen phosphorylase from Skeletal Muscle, V_max is much greater than V_max for the same enzyme from the Liver.

a) What are the physiological Functions of glycogen phosphorylase in skeletal muscle and in the liver?

b) Why must V_max be higher for the muscle form of the enzyme than for the liver enzyme?

7. Equilibrium of the reaction catalyzed by glycogen phosphorylase.

Glycogen Phosphorylase catalyzes the removal of glucose from a glycogen molecule. The ∆G′° of this reaction is 3.1 kJ/mol.

a) Determine the [P_i]/[glucose-1-phosphate] ratio for the reaction at equilibrium. (Hint: The removal of glucose from a polymeric glycogen molecule does not affect the glycogen concentration.)

b) Under physiological conditions, the [P_i]/[glucose-1-phosphate] ratio in myocytes exceeds 100:1. What does this indicate about the direction of the metabolic flux in the reaction catalyzed by glycogen phosphorylase in muscle?

c) Why do the values differ between equilibrium and physiological conditions? What is the Biological Significance of this difference?

8. Regulation of Glycogen phosphorylase.

The rate of glycogen conversion to glucose-6-phosphate in Muscle tissue is determined by The ratio of active phosphorylase a to less active phosphorylase b. Determine how the rate of glycogen breakdown will change if a muscle tissue sample containing glycogen phosphorylase is treated with a) phosphorylase kinase and ATP; b) PP1; c) epinephrine.

9. Glycogen breakdown in rabbit muscle.

The utilization of glycogen and glucose in the cell is regulated at four stages of glycolysis. To compare the Regulation of glycolysis under conditions of oxygen excess and deficiency, let us examine glucose and glycogen consumption in rabbit hindlimb Muscles in two physiological situations: at rest, which is characterized by low ATP demand, and upon the approach of the rabbit's mortal enemy, the coyote, from which the rabbit must flee to hide in its burrow. For each scenario, determine the relative levels (high, intermediate, or low) of AMP, ATP, citrate, and acetyl-CoA, as well as the effect their levels exert on the glycolytic flux regulated by specific enzymes. During moments of stress, the rabbit's leg muscles produce most of their ATP via anaerobic glycolysis (Lactic acid Fermentation) and only a small fraction through The oxidation of acetyl-CoA derived from fat breakdown.

10. Glycogen breakdown in migratory birds.

Unlike the rabbit, which hides in a burrow for a short time, migratory birds require energy reserves for prolonged flights. For example, ducks cover distances of several thousand kilometers during their annual migration. The muscles of migratory birds possess a high oxidative capacity and obtain the necessary ATP via the oxidation of acetyl-CoA (derived from fats) through The Citric Acid Cycle. Compare The regulation of glycolysis during short-term muscle activity in a fleeing rabbit with that during prolonged muscle activity in migrating ducks. Why must the regulation of glycolysis differ in these two cases?

11. Enzyme deficiencies in Carbohydrate METABOLISM.

Four clinical cases are described below. For each case, identify the defective enzyme and propose a Treatment method from those listed below. Explain your reasoning. Answer the questions provided in the description of each case. You may need to refer to the information presented in Chapter 14.

Case 1. Immediately after consuming milk, a patient experiences vomiting and diarrhea. A lactose tolerance test was performed: the patient was given a specific amount of lactose, after which blood plasma glucose and galactose concentrations were measured at regular intervals. Normally, the levels of these substances peak within an hour and then decline. In this patient, blood plasma glucose and galactose concentrations did not increase. Explain why the blood concentrations of these sugars initially rise and then fall during the test in healthy individuals. Why does this not occur in the patient?

Case 2. After consuming milk, a patient experiences vomiting and diarrhea. The patient's blood glucose concentration is low, but the concentration of reducing sugars is significantly higher than normal. Galactose is detected in the urine. Why is the blood concentration of reducing sugars elevated? Why does galactose appear in the urine?

Case 3. Following strenuous physical exertion, a patient experiences muscle cramps, but otherwise feels healthy. Muscle biopsy reveals an elevated glycogen concentration. Why does glycogen accumulation occur?

Case 4. The patient is lethargic and apathetic. The liver is enlarged, and a liver biopsy reveals an elevated content of

glycogen. The patient has a lowered Blood Glucose Level. What is the cause of the decreased blood glucose concentration in this patient?

Defective enzymes

a) Muscle phosphofructokinase-1

b) Phosphomannoisomerase

c) Galactose-1-phosphate uridylyltransferase

d) Liver phosphorylase

e) Triokinase

f) Lactase in the intestinal mucosa

g) Maltase in the intestinal mucosa

h) Muscle enzyme that cleaves bonds at glycogen branching points

Possible treatment

1. Daily 5 km jog

2. Low-fat diet

3. Low-lactose diet

4. Avoidance of heavy physical exertion

5. High doses of niacin (a precursor of NAD⁺)

6. Frequent, regular intake of small amounts of regular food

12. Manifestations of Insulin deficiency in a diabetic patient.

A person with non-insulin-dependent diabetes was admitted to the hospital in a near-comatose state. While on vacation in a remote Location, he lost his medication and had not taken insulin for two days.

a) For each of the tissues listed below, indicate whether the specific metabolic conversion in this situation is faster, slower, or the same as it would be in the same person receiving regular insulin injections.

b) For each metabolic pathway, describe at least one regulatory mechanism responsible for the changes you predicted.

Tissues and metabolic pathways

1. Adipose tissue: fatty acid synthesis

2. Muscle tissue: glycolysis, fatty acid synthesis, glycogen synthesis

3. Liver: glycolysis, Gluconeogenesis, glycogen synthesis, fatty acid synthesis, Pentose Phosphate Pathway

13. Blood metabolite levels in insulin deficiency.

Predict the levels of the following blood metabolites in the patient described in Problem 12 prior to hospital treatment, compared to the levels of these

same metabolites under normal medication conditions: (a) glucose; (b) Ketone Bodies; (c) free Fatty acids.

14. Effect of Enzyme Mutation on Metabolism.

Predict and explain the effect of each mutation-induced defect on Glycogen Metabolism: (a) loss of the cAMP-binding site on the regulatory subunit of protein kinase A (PKA); (b) absence of the protein phosphatase inhibitor (Inhibitor 1 in Fig. 15-40); (c) increased expression level of phosphorylase kinase b in the liver; (d) liver Glucagon receptor defect.

15. Hormonal Control of Metabolic Pathways.

Between dinner and breakfast, blood glucose levels drop, and the liver begins to produce rather than consume glucose. Describe the hormonal basis of these processes and explain how hormonal changes trigger glucose synthesis in the liver.

16. Metabolism in Genetically Modified Mice.

Scientists can manipulate mouse genes so that a specific Gene in a specific tissue encodes either an inactive protein (a so-called "knockout" mouse with a "disrupted" gene) or a constitutively active protein. What metabolic changes would occur in mice with the following genetic modifications: (a) knockout of the glycogen debranching enzyme in the liver; (b) liver hexokinase IV knockout; (c) liver fructose-1,6-bisphosphatase-2 (FBPase-2) knockout; (d) constitutive activity of FBPase-2 in the liver; (e) constitutive activity of AMP-activated protein kinase in muscles; (f) constitutive activity of the ChREBP protein in the liver?

Analyzing Experimental Data

17. Structure of Glycogen.

During physical exertion, muscle cells require a rapid and abundant supply of glucose. Glucose is stored in The Liver and skeletal muscles in a polymeric form as glycogen granules. Typically, a glycogen granule contains about 55,000 glucose residues (see Fig. 15-33b). Meléndez-Hevia, Waddell, and Shelton in 1993 examined several Theoretical Aspects of glycogen structure, which are explored in this problem.

a) The glycogen concentration in liver cells is approximately 0.01 M. What would the concentration of free glucose in the cells need to be to match this amount? Why is such a high concentration of free glucose problematic for the cell?

Glucose is released from glycogen by the enzyme glycogen phosphorylase, which cleaves off one glucose residue at a time from the nonreducing end of the glycogen chain. Glycogen chains are branched (see Figs. 15-26 and 15-33b), and the degree of branching (i.e., the number of branch points per chain) strongly affects the rate of glucose release.

b) Why does the rate of glucose release decrease when the degree of branching is too low (below the optimal level)? (Hint: Consider the extreme case of an unbranched chain of 55,000 glucose residues.)

c) Why does the rate of glucose release also decrease when the degree of branching is too high? (Hint: Consider physical constraints.)

Meléndez-Hevia and colleagues performed a series of calculations and found that the optimal degree of branching is two branch points per glycogen chain (see Fig. 15-33b). This is precisely the structure observed in muscle and liver glycogen.

To determine the optimal number of glucose units in a glycogen chain, two key parameters defining The structure of a glycogen granule were considered: t, the number of glucose chain tiers (layers) in the granule (the molecule in Fig. 15-33b has five such tiers), and g_c, the number of glucose residues in each chain. An attempt was made to find the values of t and g_c that maximize the following parameters: (1) The amount of glucose in the glycogen granule (G_T) per unit volume; (2) the number of unbranched glucose chains (C_A) per unit volume (i.e., the number of chains in the outer tier, readily accessible to glycogen phosphorylase); and (3) the amount of glucose accessible to phosphorylase within these unbranched chains (G_PT).

d) Show that C_A = 2^t-1. This represents the number of chains accessible to glycogen phosphorylase before the action of the debranching enzyme.

e) Show that C_T (the total number of chains in the granule) is given by the equation C_T = 2^t — 1. Thus, G_T = g_c(C_T) = g_c(2^t — 1) defines the total number of glucose residues in a glycogen granule.

f) Glycogen phosphorylase cannot remove glucose residues from glycogen chains containing fewer than five glucose residues. Show that G_PT = g_c — 4)(2^t-1). This is the amount of glucose accessible to glycogen phosphorylase.

g) Based on the size of a glucose residue and the arrangement of branches, the thickness of a single tier of glycogen chains is 0.12g_c + 0.35 nm. Show that the volume of a glycogen granule (V_s) is given by the equation: V_s = ⁴/₃ • πt³(0.12g_c + 0.35)³ nm³.

Next, the optimal values of t and g_c were determined—specifically, those values that maximize the quality function f, in which C_T, C_A, and C_PT are maximized while V_s is minimized: f = (C_TC_AC_PT)/V_s. It was found that the optimal value of g_c is independent of t.

h) Choose t in the range from 5 to 15 and find the optimal value of g_c. Compare the calculated value of g_c with that of liver glycogen (see Fig. 15-33b). (Hint: You may use spreadsheet software.)