SET PRACTICE 2 Glycolysis and glycogen breakdown 1. In working skeletal muscle under anaerobic conditions, glyceraldehyde-3-phosphate is converted into pyruvate (the payoff phase of glycolysis), and pyruvate is reduced to lactate. Write balanced equations for all of the reactions in this process, with the standard free-energy change for each (obtain from text). Then write the overall or net equation for the payoff phase of glycolysis (with lactate as the end product), including the net standard free-energy change. A "pulse-chase" experiment using 14C -labeled carbon sources is 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 the yeast extract just long enough for each intermediate in the pathway to become labeled. The label is then "chased" through the pathway by the addition of excess unlabeled glucose. The "chase" effectively 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 of 14C in the product ethanol? Explain. (b) Where would 14C have to be located in the starting glucose molecule in order to assure that all the 14C activity were liberated as 14CO2 during fermentation to ethanol? Explain.
14
2.
3.
C-Labeled glyceraldehyde-3-phosphate was added to a yeast extract. 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 label in the starting glyceraldehyde-3-phosphate? Where did the second 14 C label in fructose-1,6-bisphosphate come from? Explain.
4.
Suppose you discovered a mutant yeast whose glycolytic pathway was shorter because of the presence of a new enzyme catalyzing the reaction Glyceraldehyde-3-phosphate + H2O + NAD+ 3-phosphoglycerate + NADH + H+ Although this mutant enzyme shortens glycolysis by one step, how would it affect anaerobic ATP production? Aerobic ATP production?
5.
During strenuous activity, muscle tissue demands vast quantities of ATP compared with resting tissue. In rabbit leg muscle or turkey flight muscle, this ATP is produced almost exclusively by lactate fermentation. ATP is produced in the payoff phase of glycolysis
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by two enzymatic reactions, promoted by phosphoglycerate kinase and pyruvate kinase. 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?
6.
The oxidation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate, catalyzed by glyceraldehyde-3-phosphate dehydrogenase, proceeds with an unfavorable equilibrium constant (K'eq = 0.08; Go' = + 6.3 kJ/mol). Despite this unfavorable equilibrium, the flow through this point in the pathway proceeds smoothly. How does the cell overcome the unfavorable equilibrium?
7.
Measuring the concentrations of metabolic intermediates in the living cell presents a difficult experimental problem. Because cellular enzymes rapidly catalyze metabolic interconversions, a common problem associated with perturbing the cell experimentally is that the measured concentrations of metabolites reflect not the physiological concentrations but the equilibrium concentrations. Hence, a reliable experimental technique requires all enzyme-catalyzed reactions to be instantaneously stopped in the intact tissue, so that the metabolic intermediates do not undergo change. This objective is accomplished by rapidly compressing the tissue between large aluminum plates cooled with liquid nitrogen (-190oC), a process called freeze-clamping. After freezing, which stops enzyme action instantly, the tissue is powdered and the enzymes are inactivated by precipitation with perchloric acid. The precipitate is removed by centrifugation, and the clear supernatant extract is analyzed for metabolites. To calculate the actual intracellular concentration of the metabolite in the cell, the intracellular volume is determined from the total water content of the tissue and a measurement of the extracellular volume. The actual intracellular concentrations of the substrates and products involved in the phosphorylation of fructose-6-phosphate by the enzyme phosphofructokinase-1 in isolated rat heart tissue are given in the table below.
Metabolite Fructose-6-phosphate Fructose-1,6-bisphosphate ATP ADP
Apparent concentration (mM)* 0.087 0.022 11.42 1.32
Source: From Williamson, (1965) J.R. Glycolytic control mechanisms I. Inhibition of glycolysis by acetate and pyruvate in the isolated, perfused rat heart. J. Biol. Chem. 240, 23082321. *Calculated as mol/mL of intracellular water.
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(a) Using the information in the table, calculate the ratio, [fructose-1,6-bisphosphate] [ADP]/[fructose-6-phosphate] [ATP], for the phosphofructokinase-1 reaction under physiological conditions. (b) Given that Go' for the PFK-1 reaction is 14.2 kJ/mol, calculate the equilibrium constant for this reaction. (c) Compare the values of the mass-action ratio and K'eq. Is the physiological reaction at equilibrium? Explain.
8.
The regulated steps of glycolysis in intact cells are identified by studying the catabolism of glucose in whole tissues or organs. For example, the consumption of glucose 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 continues at the new, lower rate. Why?
9.
Glycogen phosphorylase catalyzes the removal of glucose from glycogen. Given that Go' for this reaction is 3.1 kJ/mol, calculate the ratio of [Pi] to [glucose-1-phosphate] when this reaction is at equilibrium. (Hint: The removal of glucose units from glycogen does not change the glucose units from glycogen does not change the glycogen concentration.) The measured ratio of [Pi] to [glucose-1-phosphate] in muscle cells under physiological conditions is more than 100 to 1. What does this indicate about the direction of metabolite flow through the glycogen phosphorylase reaction? Why are the equilibrium and physiological ratios different? What is the possible significance of this difference?
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Answer Key 1. G-3-P + Pi + NAD 1,3 Bis PGA + ADP 3PGA 2PGA PEP + ADP Pyruvate + NADH + H+ Net reaction: G-3-P + 2ADP + Pi
+
NADH + H + 1,3bis PGA 3PGA + ATP 2PGA PEP + H2O pyruvate + ATP lactate + NAD+
+
Go' 6.3 -18.9 4.4 1.8 -31.7 -25.2
lactate + 2ATP + H2O Go'= -63.3 kJ/mol
a.
The labeled C is on the C2 of ethanol
14
CHO OH H OH CH2 OH
14
H HO H H
14CH OPO 22 3
O HC H OH
-O
C
O CH2 OH O
14CH 3
O
CH2OPO3 2-
14CH 3
OH CH2 OH
2.
b. Work backwards from 14CO2:
CH2 OPO32-
O
14
CHO
O 14C
O
C
O
14
O H OH CH2OH HO
H
+ C O CH3 CH3
O
H
14 14
H OH OH
or
14
O
H H
C OH CH2 OH
H
CH2 OPO32-
Radiolabeled carbons could be either #3 or #4 of GLC
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3. The label has to be in position C#1 of G-3-P. This carbon derived from C4 of fructose1,6bisP. Label in position C3 comes from the fact that some of the G-3-P has to isomerizes to DHAP to create fructose-1,6bisP. The DHAP will contribute radiolabel at position C3.
4. Normally, the steps are G-3-P to 1,3bisPGA to 3PGA. 2 ATP per glucose are produced during 1,3bisPGA to 3PGA conversion. Uncoupling this step from ATP production gives a net yield of 0 ATP for anaerobic respiration. Since aerobic respiration produces much more ATP, the loss of 2 from glycolysis is much less significant.
5. No, without it, there would be no way to regenerate NAD+ and glycolysis would stop.
6. The reaction 1,3bisPGA to 3PGA is very energetically favorable and removes the 1,3bisPGA so that G is negative.
7. a. [fructose-1,6-bisphosphate] [ADP]/[fructose-6-phosphate] [ATP] =0.0292 b. Go'= -RT ln K'eq , Keq = 308 -14,200=-8.315(298) ln Keq
c. not near equilibrium, actual mass-action ratio is much less than Keq. Reaction not at equilibrium, probably by low activity of enzyme. Good place for regulation.
8. The yield of ATP/per glucose metabolized is greater in the presence of oxygen. The total amount of glucose consumes drops, because the needed amount of ATP is obtained from less glucose. Glucose consumption reaches a new steady state based on energy yield from the oxidation of the carbons all the way to carbon dioxide. 9. Reaction is Glycogen + Pi glycogen + G-1-P R=8.315 J/moloK T=298oK Go'= -RT ln K'eq 3100= 8.315 (298) (ln Keq) Keq= 0.286= [G-1-P] [Pi] Problem asks to compare the reciprocal of this: [Pi] . [G-1-P]
Go'= 3.1 kJ/mol
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or 1/0.286 = 3.5 to the observed ratio of >100. G-1-P is at lower concentration than equilibrium concentration by conversion to G-6-P. This results in a negative G. G = Go' + RT ln [products ] [reactants] G = 3,100 + 8.315 (298) ln .01 G = 3,100 + -11,411 G = -8,311
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