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Lehninger Principles of Biochemistry

David L. Nelson, Michael M. Cox

Chapter 16

Citric Acid Cycle - all with Video Answers

Educators

Chapter Questions

13:06

Problem 1

Balance Sheet for the Citric Acid Cycle The citric acid cycle has eight enzymes: citrate synthase, aconitase, isocitrate dehydrogenase, $\alpha$-ketoglutarate dehydrogenase, succinyl-CoA synthetase, succinate dehydrogenase, fumarase, and malate dehydrogenase.
(a) Write a balanced equation for the reaction catalyzed by each enzyme.
(b) Name the cofactor(s) required by each enzyme reaction.
(c) 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 $\mathrm{CO}_2$ ); oxidation-reduction; substrate-level phosphorylation; isomerization.
(d) Write a balanced net equation for the catabolism of acetyl-CoA to $\mathrm{CO}_2$.

Shazia Naz
Shazia Naz
Numerade Educator
05:13

Problem 2

Recognizing Oxidation and Reduction Reactions One biochemical strategy of many living organisms is the stepwise oxidation of organic compounds to $\mathrm{CO}_2$ and $\mathrm{H}_2 \mathrm{O}$ and the conservation of a major part of the energy thus produced in the form of ATP. It is important to be able to recognize oxidation-reduction processes in metabolism. Reduction of an organic molecule results from the hydrogenation of a double bond (Eqn 1, below) or of a single bond with accompanying cleavage (Eqn 2). Conversely, oxidation results from dehydrogenation. In biochemical redox reactions, the coenzymes NAD and FAD dehydrogenate/hydrogenate organic molecules in the presence of the proper enzymes.
For each of the metabolic transformations in (a) through (h), determine whether oxidation or reduction has occurred. Balance each transformation by inserting $\mathrm{H}-\mathrm{H}$ and, where necessary, $\mathrm{H}_2 \mathrm{O}$.
a.figure cant copy
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Shazia Naz
Shazia Naz
Numerade Educator
03:34

Problem 3

Relationship between Energy Release and the Oxidation State of Carbon A eukaryotic cell can use glucose $\left(\mathrm{C}_6 \mathrm{H}_{12} \mathrm{O}_6\right)$ and hexanoic acid $\left(\mathrm{C}_6 \mathrm{H}_{14} \mathrm{O}_2\right)$ as fuels for cellular respiration. On the basis of their structural formulas, which substance releases more energy per gram on complete combustion to $\mathrm{CO}_2$ and $\mathrm{H}_2 \mathrm{O}$ ?

Shazia Naz
Shazia Naz
Numerade Educator
03:32

Problem 4

Nicotinamide Coenzymes as Reversible Redox Carriers The nicotinamide coenzymes (see Fig. 13-15) can undergo reversible oxidation-reduction reactions with specific substrates in the presence of the appropriate dehydrogenase. In these reactions, $\mathrm{NADH}+\mathrm{H}^{+}$serves as the hydrogen source, as described in Problem 2. Whenever the coenzyme is oxidized, a substrate must be simultaneously reduced:
$$
\underset{\text { Oxidized }}{\text { Substrate }}+\underset{\text { Reduced }}{\mathrm{NADH}}+\mathrm{H}^{+} \rightleftharpoons \underset{\text { Reduced }}{\text { product }}+\underset{\text { Oxidized }}{\mathrm{NAD}^{+}}
$$

For each of the reactions in (a) through (f), determine whether the substrate has been oxidized or reduced or is unchanged in oxidation state (see Problem 2). If a redox change has occurred, balance the reaction with the necessary amount of $\mathrm{NAD}^{+}, \mathrm{NADH}, \mathrm{H}^{+}$, and $\mathrm{H}_2 \mathrm{O}$. The objective is to recognize when a redox coenzyme is necessary in a metabolic reaction.
a.figure cant copy
b.figure cant copy
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Shazia Naz
Shazia Naz
Numerade Educator
02:42

Problem 5

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 $\mathrm{CO}_2$ and $\mathrm{H}_2 \mathrm{O}$ ) 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?

Pronoy Sinha
Pronoy Sinha
Numerade Educator
04:29

Problem 6

Formation of Oxaloacetate in a Mitochondrion In the last reaction of the citric acid cycle, malate is dehydrogenated to regenerate the oxaloacetate necessary for the entry of acetyl-CoA into the cycle:
$$
\begin{aligned}
\text { L-Malate }+\mathrm{NAD}^{+} \longrightarrow \text { oxaloacetate }+\mathrm{NADH}+\mathrm{H}^{+} \\
\Delta G^{\prime 0}=30.0 \mathrm{~kJ} / \mathrm{mol}
\end{aligned}
$$
(a) Calculate the equilibrium constant for this reaction at $25^{\circ} \mathrm{C}$.
(b) Because $\Delta G^{\prime 0}$ assumes a standard pH of 7 , the equilibrium constant calculated in (a) corresponds to
$$
K_{\text {eq }}^{\prime}=\frac{[\text { oxaloacetate }][\mathrm{NADH}]}{[\text { L-malate }]\left[\mathrm{NAD}^{+}\right]}
$$

The measured concentration of l-malate in rat liver mitochondria is about 0.20 mm when $\left[\mathrm{NAD}^{+}\right] /[\mathrm{NADH}]$ is 10 . Calculate the concentration of oxaloacetate at pH 7 in these mitochondria.
(c) To appreciate the magnitude of the mitochondrial oxaloacetate concentration, calculate the number of oxaloacetate molecules in a single rat liver mitochondrion. Assume the mitochondrion is a sphere of diameter $2.0 \mu \mathrm{m}$.

Sana Riaz
Sana Riaz
Numerade Educator
02:40

Problem 7

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

Pronoy Sinha
Pronoy Sinha
Numerade Educator
09:17

Problem 8

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 source, respiration soon stops and a metabolic intermediate accumulates.
(a) What is the structure of this intermediate?
(b) Explain why it accumulates.
(c) Explain why oxygen consumption stops.
(d) Aside from removal of the malonate, how can this inhibition of respiration be overcome? Explain.

Sana Riaz
Sana Riaz
Numerade Educator
05:15

Problem 9

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 $\mathrm{CO}_2$ and $\mathrm{H}_2 \mathrm{O}$ ?
(b) Suppose you add a brief pulse of [3- $\left.{ }^{14} \mathrm{C}\right]$ pyruvate (labeled in the methyl position) to the mitochondria. After one turn of the citric acid cycle, what is the location of the ${ }^{14} \mathrm{C}$ in the oxaloacetate? Explain by tracing the ${ }^{14} \mathrm{C}$ label through the pathway. How many turns of the cycle are required to release all the $\left[3-{ }^{14} \mathrm{C}\right]$ pyruvate as $\mathrm{CO}_2$ ?

Sana Riaz
Sana Riaz
Numerade Educator
04:05

Problem 10

$\left[1-{ }^{14} \mathrm{C}\right]$ Glucose Catabolism An actively respiring bacterial culture is briefly incubated with $\left[1-^{14} \mathrm{C}\right]$ glucose, and the glycolytic and citric acid cycle intermediates are isolated. Where is the ${ }^{14} \mathrm{C}$ in each of the intermediates listed below? Consider only the initial incorporation of ${ }^{14} \mathrm{C}$, 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) $\alpha$-Ketoglutarate
(g) Oxaloacetate

Sana Riaz
Sana Riaz
Numerade Educator
01:45

Problem 11

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

Pronoy Sinha
Pronoy Sinha
Numerade Educator
05:05

Problem 12

Synthesis of Oxaloacetate by the Citric Acid Cycle Oxaloacetate is formed in the last step of the citric acid cycle by the $\mathrm{NAD}^{+}$-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?

Sana Riaz
Sana Riaz
Numerade Educator
09:45

Problem 13

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:
$$
\begin{aligned}
& \mathrm{F}-\mathrm{CH}_2 \mathrm{COO}^{-}+\mathrm{CoA}-\mathrm{SH}+\mathrm{ATP} \longrightarrow \\
& \underset{\|}{\mathrm{F}}-\mathrm{CH}_2 \mathrm{C}-\mathrm{S}-\mathrm{CoA}+\mathrm{AMP}+\mathrm{PP}_1 \\
&
\end{aligned}
$$

The toxic effect of fluoroacetate was studied in an experiment using intact isolated rat heart. After the heart was perfused with 0.22 mm fluoroacetate, the measured rate of glucose uptake and glycolysis decreased, and glucose 6-phosphate and fructose 6-phosphate accumulated. Examination of the citric acid cycle intermediates revealed that their concentrations were below normal, except for citrate, with a concentration 10 times higher than normal.
(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) Fluoroacetyl-CoA is enzymatically transformed in the citric acid cycle. What is the structure of the end product of fluoroacetate metabolism? Why does it block the citric acid cycle? How might the inhibition be overcome?
(c) In the heart perfusion experiments, why did glucose uptake and glycolysis decrease? Why did hexose monophosphates accumulate?
(d) Why is fluoroacetate poisoning fatal?

Ronald Prasad
Ronald Prasad
Numerade Educator
04:07

Problem 14

Synthesis of L-Malate in Wine Making The tartness of some wines is due to high concentrations of L-malate. Write a sequence of reactions showing how yeast cells synthesize L-malate from glucose under anaerobic conditions in the presence of dissolved $\mathrm{CO}_2\left(\mathrm{HCO}_3^{-}\right)$. Note that the overall reaction for this fermentation cannot involve the consumption of nicotinamide coenzymes or citric acid cycle intermediates.

Pronoy Sinha
Pronoy Sinha
Numerade Educator
04:46

Problem 15

Net Synthesis of $\boldsymbol{\alpha}$-Ketoglutarate $\alpha$-Ketoglutarate plays a central role in the biosynthesis of several amino acids. Write a sequence of enzymatic reactions that could result in the net synthesis of $\alpha$-ketoglutarate from pyruvate. Your proposed sequence must not involve the net consumption of other citric acid cycle intermediates. Write an equation for the overall reaction and identify the source of each reactant.

Pronoy Sinha
Pronoy Sinha
Numerade Educator
07:25

Problem 16

Regulation of the Pyruvate Dehydrogenase Complex In animal tissues, the rate of conversion of pyruvate to acetyl-CoA is regulated by the ratio of active, phosphorylated to inactive, unphosphorylated PDH complex. Determine what happens to the rate of this reaction when a preparation of rabbit muscle mitochondria containing the PDH complex is treated with (a) pyruvate dehydrogenase kinase, ATP, and NADH ; (b) pyruvate dehydrogenase phosphatase and $\mathrm{Ca}^{2+}$; (c) malonate.

Bryan Valdivia
Bryan Valdivia
Numerade Educator
03:48

Problem 17

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 $\mathrm{FeCl}_3$ in the culture medium, as indicated in the graph. Why does the yield decrease when the concentration of $\mathrm{Fe}^{3+}$ is above or below the optimal value of $0.5 \mathrm{mg} / \mathrm{L}$ ?
(b) Write the sequence of reactions by which A. niger synthesizes citric acid from sucrose. Write an equation for the overall reaction.
(c) Does the commercial process require the culture medium to be aerated-that is, is this a fermentation or an aerobic process? Explain.

Sana Riaz
Sana Riaz
Numerade Educator
02:54

Problem 18

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. 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. 6-29.) Why is succinyl-CoA an appropriate signal for regulation of the citric acid cycle? How does the regulation of citrate synthase control the rate of cellular respiration in pig heart tissue?

Prashant Bana
Prashant Bana
Numerade Educator
04:13

Problem 19

Regulation of Pyruvate Carboxylase The carboxylation of pyruvate by pyruvate carboxylase occurs at a very low rate unless acetyl-CoA, a positive allosteric modulator, is present. If you have just eaten a meal rich in fatty acids (triacylglycerols) but low in carbohydrates (glucose), how does this regulatory property shut down the oxidation of glucose to $\mathrm{CO}_2$ and $\mathrm{H}_2 \mathrm{O}$ but increase the oxidation of acetyl-CoA derived from fatty acids?

Ronald Prasad
Ronald Prasad
Numerade Educator
01:49

Problem 20

Relationship between Respiration and the Citric Acid Cycle Although oxygen does not participate directly in the citric acid cycle, the cycle operates only when $\mathrm{O}_2$ is present. Why?

Pronoy Sinha
Pronoy Sinha
Numerade Educator
07:12

Problem 21

Thermodynamics of Citrate Synthase Reaction in Cells Citrate is formed by the condensation of acetyl-CoA with oxaloacetate, catalyzed by citrate synthase:
$$
\text { Oxaloacetate }+ \text { acetyl-CoA }+\mathrm{H}_2 \mathrm{O} \longrightarrow \text { citrate }+\mathrm{CoA}+\mathrm{H}^{+}
$$

In rat heart mitochondria at pH 7.0 and $25^{\circ} \mathrm{C}$, the concentrations of reactants and products are: oxaloacetate, $1 \mu \mathrm{M}$; acetyl-CoA, $1 \mu \mathrm{s}$; citrate, $220 \mu \mathrm{m}$; and $\mathrm{CoA}, 65 \mu \mathrm{m}$. The standard free-energy change for the citrate synthase reaction is $-32.2 \mathrm{~kJ} / \mathrm{mol}$. What is the direction of metabolite flow through the citrate synthase reaction in rat heart cells? Explain.

Sana Riaz
Sana Riaz
Numerade Educator
10:04

Problem 22

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.

Pronoy Sinha
Pronoy Sinha
Numerade Educator