Chapter 17- The Citric Acid Cycle

Question 1

Citric acid cycle

Answer 1

Also called the tricarboxylic acid (TCA) cycle or the Krebs cycle. It is the complete oxidation of glucose derivatives to carbon dioxide. The citric acid cycle is the final pathway for the oxidation of fuel molecules like carbohydrates, fatty acids, and amino acids. Under aerobic conditions, the pyruvate generated from glucose is oxidatively decarboxylated to form acetyl CoA.

Question 2

Most fuel molecules enter the citric acid cycle as

Answer 2

Acetyl coenzyme A- this is the main entry point to the cycle.

Question 3

Where do the reactions of the citric acid cycle take place?

Answer 3

The eukaryotes, the reactions take place in the matrix of the mitochondria. Pyruvate enters the mitochondria and is converted acetyl CoA

Question 4

What is the importance of the citric acid cycle to the cell? (2)

Answer 4

1. It is the gateway to the aerobic metabolism of any molecule that can be transformed into an acetyl group or a component of the citric acid cycle
2. It is an important source of precursors for the building blocks of many other molecules like amino acids, nucleotide bases, and porphyrin (the organic component of heme). Oxaloacetate is a citric acid cycle component that is an important precursor to glucose.

Question 5

Fuel molecules

Answer 5

Carbon compounds that are capable of being oxidized- of losing electrons

Question 6

What is the function of the citric acid cycle in transforming fuel molecules into ATP?

Answer 6

The citric acid cycle includes a series of oxidation-reduction reactions that result in the oxidation of an acetyl group to two molecules of carbon dioxide. The oxidation reaction generates high energy electrons that will be used to power the synthesis of ATP. The citric acid cycle harvests high energy electrons from carbon fuels

Question 7

Structure of the mitochondria

Answer 7

The mitochondria has an inner mitochondrial membrane and an outer mitochondrial membrane. The inner membrane has invaginations called cristae. The oxidative decarboxylation of pyruvate and the sequence of reactions in the citric acid cycle takes place within the matrix

Question 8

Overall pattern of the citric acid cycle

Answer 8

A 4 carbon compound (oxaloacetate) condenses with a 2 carbon acetyl unit to make a 6 carbon tricarboxylic acid (citrate). The 6 carbon compound releases carbon dioxide twice, in 2 successive oxidative decarboxylations that yield high energy electrons. A 4 carbon compound remains- it is further processed to regenerate oxaloacetate, which can initiate another round of the cycle. Overall, 2 carbon atoms enter the cycle as an acetyl unit and 2 carbon atoms leave the cycle in the form of two molecules of carbon dioxide.

Question 9

Key function of the citric acid cycle

Answer 9

To harvest high energy electrons in the form of NADH and FADH2.

Question 10

Formation of NADH and FADH2 in the citric acid cycle

Answer 10

The citric acid cycle removes electrons from acetyl CoA and uses these electrons to reduce NAD+ and FAD, forming NADH and FADH2. Six electrons (3 hydride ions) are transferred to 3 molecules of NAD+, and one pair of hydrogen atoms (2 electrons) is transferred to one molecule of FAD each time an acetyl CoA is processed by the cycle

Question 11

Electron transport chain

Answer 11

A series of membrane proteins- the electrons released in the reoxidation of NADH and FADH2 flow through the transport chain. This generates a proton gradient across the inner mitochondrial membrane. These protons then flow through ATP synthase to generate ATP from ADP and inorganic phosphate.

Question 12

Oxidative phosphorylation

Answer 12

The electron carriers from the electron transport chain yield 9 molecules of ATP when they are oxidized by oxygen in oxidative phosphorylation. This reduction of oxygen to water and the synthesis of ATP makes up oxidative phosphorylation

Question 13

Products of the citric acid cycle (3)

Answer 13

A 2 carbon unit is oxidized to make 2 molecules of carbon dioxide, one molecule of ATP, and high energy electrons in the form of NADH and FADH2

Question 14

Cellular respiration

Answer 14

The citric acid cycle constitutes the first stage in cellular respiration. The high energy electrons produced by the citric acid cycle reduce oxygen to generate a proton gradient. This proton gradient is ultimately used to synthesize ATP through oxidative phosphorylation (second stage of respiration).

Question 15

The citric acid cycle is responsible for producing what proportion of energy used by the cells?

Answer 15

In conjunction with oxidative phosphorylation, the citric acid cycle provides 90% of the energy used by human cells.

Question 16

Why is the citric acid cycle considered highly efficient?

Answer 16

The oxidation of a limited number of citric acid cycle molecules can generate large amounts of NADH and FADH2

Question 17

Oxaloacetate

Answer 17

The 4 carbon molecule that initiates the first step in the citric acid cycle. It is regenerated at the end of one passage through the cycle. Therefore, one molecule of oxaloacetate is capable of participating in the oxidation of many acetyl molecules.

Question 18

Pyruvate dehydrogenase complex

Answer 18

A complex of 3 distinct enzymes that oxidatively decarboxylates pyruvate to form acetyl CoA in the mitochondrial matrix. Acetyl CoA is the fuel needed for the citric acid cycle. Under aerobic conditions, glucose is broken down into pyruvate and transported into the mitochondria by a specific carrier protein in the mitochondrial membrane. The decarboxylation of pyruvate by the pyruvate dehydrogenase complex is an irreversible reaction that is the link between glycolysis and the citric acid cycle. The PDC produces carbon dioxide and captures high transfer potential electrons in the form of NADH

Question 19

Reactants (3) and products (4) of the pyruvate dehydrogenase complex reaction

Answer 19

Pyruvate, CoA, and NAD+ are the reactants. They yield acetyl CoA, carbon dioxide, NADH, and H+

Question 20

Pyruvate dehydrogenase complex of E. coli enzymes (3)

Answer 20

3 enzymes:
1. Pyruvate dehydrogenase component (E1)
2. Dihydrolipoyl transacetylase (E2)
3. Dihydrolipoyl dehydrogenase (E3)

Question 21

Pyruvate dehydrogenase component (E1)

Answer 21

Its prosthetic group is TPP. It catalyzes the oxidative decarboxylation of pyruvate in E. coli

Question 22

Dihydrolipoyl transacetylase (E2)

Answer 22

Its prosthetic group is lipoamide. It catalyzes the transfer of acetyl groups to CoA in E. coli

Question 23

Dihydrolipoyl dehydrogenase (E3)

Answer 23

Its prosthetic group is FAD. It catalyzes the regeneration of the oxidized form of lipoamide in E. coli.

Question 24

The synthesis of acetyl coenzyme A from pyruvate requires which enzymes?

Answer 24

It requires 3 enzymes and 5 coenzymes. These includes the 3 enzymes of the pyruvate dehydrogenase complex.

Question 25

5 coenzymes required in the synthesis of acetyl coenzyme A from pyruvate

Answer 25

1. Catalytic cofactors- thiamine pyrophosphate (TPP), lipoic acid, and FAD
2. Stoichiometric cofactors- CoA and NAD+

Question 26

What are stoichiometric cofactors?

Answer 26

Cofactors that function as substrates- includes CoA and NAD+

Question 27

Coenzymes

Answer 27

Small organic molecules that are often derived from vitamins. Coenzymes can bind loosely with the enzyme and release from the active site. Therefore, they are also considered substrates for the reaction. Alternatively, they may be tight binding and cannot dissociate easily from the enzyme. In this case, after their initial participation in an enzyme-catalyzed reaction, the enzyme would no longer be able to use the cofactor in another round of catalysis until the initial state of the cofactor is reformed, which takes another chemical reaction and often an additional substrate.

Question 28

Cofactors

Answer 28

Molecules that bind to enzymes and are required for catalytic activity. They can be divided into two major categories: metals and coenzymes. Metal cofactors commonly found in human enzymes include iron and magnesium

Question 29

4 steps of conversion of pyruvate into acetyl CoA

Answer 29

1. Decarboxylation
2. Oxidation
3. Transfer
4. Regenerates the active enzyme
   These steps must be coupled to preserve the free energy derived from the decarboxylation step to drive the formation of NADH and acetyl CoA

Question 30

Decarboxylation (acetyl CoA synthesis)

Answer 30

Pyruvate combines with the ionized form of TPP and is then decarboxylated to yield hydroxyethyl-TPP and a molecule of carbon dioxide. This reaction is catalyzed by the pyruvate dehydrogenase component (E1) of the pyruvate dehydrogenase complex. TPP is the prosthetic group of the pyruvate dehydrogenase component

Question 31

Why must the steps in the synthesis of acetyl CoA be coupled?

Answer 31

To preserve the free energy derived from the decarboxylation step, since this drives the formation of NADH and acetyl CoA.

Question 32

Decarboxylation mechanism (4)

Answer 32

In TPP, the carbon atom between the nitrogen and sulfur atoms in the thiazole ring is more acidic than is typical for those types of groups.
1. This carbon center ionizes to form a carbanion
2. The carbanion readily adds to the carbonyl group of pyruvate
3. This addition is followed by the decarboxylation of pyruvate. The positively charged ring of TPP stabilizes the negative charge that is transferred to the ring as part of the decarboxylation
4. Protonation yields a hydroxyethyl-TPP intermediate

Question 33

Oxidation (acetyl CoA synthesis)

Answer 33

Also catalyzed by the pyruvate dehydrogenase complex (E1 component). The hydroxyethyl group attached to TPP is oxidized to form an acetyl group while being simultaneously transferred to lipoamide (dihydroxylipoamide)- a derivative of lipoic acid that is linked to the side chain of a lysine residue by an amide linkage. The transfer of the hydroxyethyl group results in the formation of an energy rich thioester bond. The disulfide group of lipoamide is reduced to its disulfhydryl form. This reaction yields acetyllipoamide, formed on E2.

Question 34

Thiazole

Answer 34

5-membered heterocyclic compound that contains both sulfur and nitrogen. There are 2 double bonds, one carbon-carbon and the other nitrogen-carbon

Question 35

Carbonyl group

Answer 35

A carbon double bonded to an oxygen atom

Question 36

Formation of acetyl CoA (acetyl CoA synthesis)

Answer 36

The acetyl group is transferred from acetyllipoamide to CoA to form acetyl CoA. The E2 component of the PDC catalyzes this reaction. The energy-rich thioester bond is preserved as the acetyl group is transferred to CoA. CoA serves as a carrier of the activated acetyl group. At the end of this reaction, acetyl CoA, the fuel for the citric acid cycle, has now been generated from pyruvate

Question 37

Regeneration of oxidized lipoamide (acetyl CoA synthesis)

Answer 37

The pyruvate dehydrogenase complex won’t be able to complete another catalytic cycle until the dihydroxylipoamide is oxidized to lipoamide. In this step, the oxidized form of lipoamide is regenerated by the E3 component of the PDC. 2 electrons are transferred to an FAD prosthetic group of the enzyme and then to NAD+. This regenerates NADH

Question 38

Flavoproteins

Answer 38

Proteins tightly associated with FAD or flavin (FMN). FAD usually receives electrons from NADH, but electrons transfer from FAD to NAD+ to regenerate oxidized lipoamide. The electron-transfer potential of FAD is increased by its chemical environment within the enzyme, enabling it to transfer electrons to NAD+

Question 39

Structure of the pyruvate dehydrogenase complex

Answer 39

The three enzymes of the pyruvate dehydrogenase complex are structurally integrated, and the lipoamide arm allows rapid movement of substrates and products from one active site of the complex to another. The core of the pyruvate dehydrogenase complex is formed by 60 molecules of E2, the transacetylase.

Question 40

Transacetylase

Answer 40

The core of the PDC is formed by 60 molecules of the transacetylase component E2. Transacetylase consists of 20 catalytic trimers assembled to form a hollow cube. Each of the 3 subunits forming a trimer has 3 major domains. At the amino terminus is a small domain that contains a bound flexible lipoamide cofactor attached to a lysine residue. The lipoamide domain is followed by a small domain that interacts with E3 within the complex. A larger transacetylase domain completes an E2 subunit

Question 41

Dihydrolipoamide

Answer 41

Formed by the attachment of the vitamin lipoic acid to a lysine residue in dihydrolipoyl transacetylase (E2)

Question 42

Structure of the transacetylase (E2) core

Answer 42

Each subunit of the transacetylase trimer consists of 3 domains: a lipoamide-binding domain, a small domain that interacts with E3, and a large transacetylase catalytic domain. The catalytic domains interact with one another to form the catalytic trimer.

Question 43

How do the 3 active sites of the pyruvate dehydrogenase complex work together?

Answer 43

The long, flexible lipoamide arm of the E2 subunit carries substrates from active site to active site. All the intermediates in the oxidative decarboxylation of pyruvate remain bound to the complex throughout the reaction sequence and are readily transferred as the flexible arm of E2 calls on each active site in turn. This allows for coordinated catalysis of a complex reaction. The proximity of one enzyme to another increases the overall reaction rate and minimizes side reactions

Question 44

Steps in the pyruvate dehydrogenase mechanism (6)

Answer 44

1. Pyruvate is decarboxylated at the active site of E1, forming the hydroxyethyl-TPP intermediate, and carbon dioxide leaves (first product).
2. E2 inserts the lipoamide arm of the lipoamide domain into the deep channel in E1 leading to the active site
3. E1 catalyzes the transfer of the acetyl group to the lipoamide.
4. The acetyl moiety is then transferred to CoA, and the second product, acetyl CoA, leaves the cube. The reduced lipoamide arm then swings to the active site of the E3 flavoprotein.
5. At the E3 active site, the lipoamide is oxidized by coenzyme FAD. The reactivated lipoamide is ready to begin another reaction cycle
6. The final product, NADH, is produced with the reoxidation of FADH2 to FAD.

Question 45

Active site of E1

Answer 45

The active site of E1 of pyruvate dehydrogenase lies deep within the E1 complex, connected to the enzyme surface by a 20 A long hydrophobic channel

Question 46

Transfer of the acetyl group to the lipoamide (pyruvate dehydrogenase mechanism)

Answer 46

E1 catalyzes the transfer. The acetylated arm then leaves E1 and enters the E2 cube to visit the active site of E2, located deep in the cube at the subunit interface

Question 47

Citrate synthase

Answer 47

Catalyzes the condensation of oxaloacetate (4 carbon unit) and the acetyl group of acetyl CoA (2 carbon unit). It catalyzes this reaction by bringing the substrates into close proximity, orienting them, and polarizing certain bonds.

Question 48

Citrate synthase reaction

Answer 48

Oxaloacetate reacts with acetyl CoA and water to yield citrate and CoA. This is reaction is an aldol condensation followed by a hydrolysis. Oxaloacetate first condenses with acetyl CoA to form citryl CoA- this molecule is energy rich because it contains the thioester bond (which originated in acetyl CoA). The hydrolysis of citryl CoA thioester to citrate powers the synthesis of the new molecules, which is citrate. It drives what would otherwise be a relatively unfavorable lengthening of the carbon chain

Question 49

Synthase

Answer 49

An enzyme catalyzing a synthetic reaction in which 2 units are joined usually without the direct participation of ATP or another nucleoside triphosphate

Question 50

How does the mechanism of citrate synthase prevent undesirable reactions?

Answer 50

The condensation of acetyl CoA and oxaloacetate initiates the citric acid cycle, so it’s important that side reactions are minimized. There is ordered binding, because when oxaloacetate binds, citrate synthase undergoes conformational changes to fit the enzyme (induced fit).

Question 51

Condensation reaction

Answer 51

A reaction in which two molecules combine to form a single molecule. A small molecule, often water, is usually removed during a condensation reaction.

Question 52

Ordered kinetics of citrate synthase

Answer 52

Citrate synthase exhibits sequential, ordered kinetics- oxaloacetate binds first, followed by acetyl CoA. Ordered binding is necessary because oxaloacetate induces a major structural rearrangement to make a binding site for acetyl CoA- this is induced fit

Question 53

Citrate synthase induced fit

Answer 53

When oxaloacetate binds, the enzyme converts to a more closed form. In each subunit, the small domain rotates slightly to rotate the alpha helices around the bound oxaloacetate. The structural changes create a binding site for acetyl CoA. During the reaction additional structural changes in the enzyme cause the active site to become completely enclosed.

Question 54

Mechanism of synthesis of citryl CoA by citrate synthase (4)

Answer 54

1. In the substrate complex, His 274 donates a proton to the carbonyl oxygen of acetyl CoA to promote the removal of a methyl proton by Asp 375 to form the enol intermediate
2. Oxaloacetate is activated by the transfer of a proton from His 320 to its carbonyl carbon.
3. Simultaneously, the enol of acetyl CoA attacks the carbonyl carbon of oxaloacetate to form a carbon-carbon bond linking acetyl CoA and oxaloacetate. Citryl CoA is formed.
4. His 274 acts as a proton donor to hydrolyze the thioester, which yields citrate and CoA

Question 55

Aconitase

Answer 55

An iron-sulfur protein (it contains iron that is not bonded to heme). Its 4 iron atoms are complexed to 4 inorganic sulfides and 3 cysteine sulfur atoms, forming its active site. There is one iron atom available to bind citrate through one of its COO- groups and the OH group of citrate. This Fe-S cluster participates in dehydrating and rehydrating the bound substrate. The aconitase enzyme catalyzes both the dehydration and hydration steps in the isomerization of citrate.

Question 56

Why is citrate isomerized?

Answer 56

The hydroxyl group is not properly located in the citrate molecule for the oxidative decarboxylations that follow. Therefore, citrate needs to be isomerized into isocitrate to allow the 6 carbon unit to undergo oxidative decarboxylation. The isomerization includes a dehydration step followed by a hydration step. This allows for the interchange of H and OH. Both of the steps are catalyzed by aconitase

Question 57

Isocitrate dehydrogenase

Answer 57

Catalyzes the oxidative decarboxylation of isocitrate. It forms an unstable intermediate called oxalosuccinate during the reaction. While it is bound to the enzyme, it loses carbon dioxide to form alpha ketoglutarate. The first 2 carbon dioxide molecules produced during the citric acid cycle are lost during this reaction. The oxidation reaction forms the electron carrier NADH.

Question 58

α-ketoglutarate dehydrogenase complex

Answer 58

After isocitrate is converted into alpha ketoglutarate, a second oxidative decarboxylation reaction occurs. The alpha-ketoglutarate is used to make succinyl CoA, and the alpha-ketoglutarate dehydrogenase complex catalyzes the reaction to synthesis succinyl CoA. This complex is an organized assembly of 3 kinds of enzymes. The reaction includes the decarboxylation of an alpha-ketoacid and the subsequent formation of a thioester linkage with CoA that has high transfer potential. NADH is produced

Question 59

How is the α-ketoglutarate dehydrogenase complex similar to the pyruvate dehydrogenase complex?

Answer 59

The α-ketoglutarate dehydrogenase complex is an organized assembly of 3 kinds of enzymes that is homologous to the pyruvate dehydrogenase complex. The E3 component is identical in both enzymes. The oxidative decarboxylation of α-ketoglutarate closely resembles that of pyruvate- both compounds are alpha-ketoacids. Both reactions include the decarboxylation of an alpha ketoacid and the formation of a thioester linkage with CoA. The reaction mechanisms are analogous.

Question 60

Succinyl CoA synthetase

Answer 60

Catalyzes the cleavage of a
thioester linkage and simultaneously forms ATP. It cleaves the thioester bond to power the synthesis of the 6 carbon citrate from the 4 carbon oxaloacetate and the 2 carbon fragment. The cleavage of the thioester bond of succinyl CoA is coupled to the phosphorylation of a purine nucleoside diphosphate like ADP. It is the only step in the citric acid cycle that directly yields a compound with high phosphoryl transfer potential

Question 61

Isozymes of succinyl CoA synthetase

Answer 61

In mammals, there are two isozymes, one specific for ADP and one specific for GDP. In tissues like the skeletal and heart muscle, which perform large amounts of cellular respiration, the ADP-requiring isozyme predominates. The GDP-requiring isozyme is common is tissues the perform many anabolic reactions, like the liver. The GDP enzyme seems to work in the opposite direction of the TCA cycle- GTP is used to power the synthesis of succinyl CoA, which is a precursor for heme synthesis

Question 62

Substrate-level phosphorylation and succinyl CoA

Answer 62

The formation of ATP at the expense of succinyl CoA is an example of substrate-level phosphorylation. This is because succinyl phosphate, a high phosphoryl-transfer potential
compound, donates a phosphate to ADP. A histidine residue acts as a moving arm that detaches the phosphoryl group. It then swings over to a bound ADP and transfers the phosphoryl group to form ATP.

Question 63

Nucleoside diphosphokinase

Answer 63

Interconverts between nucleotide triphosphates if one NTP accumulates but the other is needed. ADP and GDP are each more commonly needed in different tissues

Question 64

Succinyl coenzyme A synthetase-catalyzed reaction mechanism (4 steps)

Answer 64

1. Orthophosphate displaces coenzyme A, which generates another energy-rich compound, succinyl phosphate
2. A histidine residue removes the phosphoryl group with succinate and phosphohistidine being generated simultaneously
3. The phosphohistidine residue then swings over to a bound ADP
4. The phosphoryl group is transferred to form ATP

Question 65

Oxidation of succinate

Answer 65

The final stage of the citric acid cycle, which regenerates oxaloacetate. A methylene group (CH2) is converted into a carbonyl group (C=O) in 3 reactions- oxidation, hydration, and a second oxidation reaction. Oxaloacetate is regenerated for another round of the cycle, and more energy is extracted in the form of FADH2 and NADH. Oxaloacetate can condense with another acetyl CoA
to initiate another cycle of the citric acid cycle

Question 66

Succinate dehydrogenase

Answer 66

Oxidizes succinate to fumarate. FAD is used as a hydrogen acceptor in this reaction because NAD+ can’t be used- the free energy change is insufficient to reduce NAD+ and FAD is generally used to remove two hydrogen atoms from a substrate. Succinate dehydrogenase is an iron-sulfur protein. The FADH2 produced by the oxidation of succinate does not dissociate from the enzyme- 2 electrons are transferred directly from FADH2 to iron-sulfur clusters of the enzyme, which passes the electrons directly to coenzyme Q

Question 67

3 enzymes required to regenerate oxaloacetate

Answer 67

Succinate dehydrogenase, fumarase, and malate
dehydrogenase

Question 68

Succinate dehydrogenase location

Answer 68

It is different from other enzymes in the citric acid cycle because it’s embedded in the inner mitochondrial membrane. Succinate dehydrogenase is directly associated with the electron transport chain, which is the link between the citric acid cycle and ATP formation

Question 69

Coenzyme Q (CoQ)

Answer 69

Receives electrons directly from FADH2 in the oxidation of succinate. It is an important member of the electron transport chain, and passes electrons to the ultimate electron acceptor (molecular oxygen).

Question 70

Fumarase

Answer 70

Catalyzes the reaction in the regeneration of oxaloacetate where fumarate is hydrated to form L-malate. It catalyzes a stereospecific trans addition of H+ and OH-. The OH- group adds to only one side of the double bond of fumarate. Therefore, only an L isomer of malate is formed.

Question 71

Malate dehydrogenase

Answer 71

Catalyzes the oxidation of malate to form oxaloacetate. NAD+ acts as the hydrogen acceptor, producing a molecule of NADH. This reaction is extremely endergonic under standard
conditions. The actual ΔG makes the reaction favorable because of the use of the products: oxaloacetate in the first step of the citric acid cycle and NADH in the electron transport chain

Question 72

Regeneration of oxaloacetate (3 steps)

Answer 72

1. Succinate is oxidized to form fumarate by succinate dehydrogenase. Produces FAD2.
2. Fumarate is hydrated by fumarase (using one molecule of water) to form L-malate
3. Malate is oxidized to form oxaloacetate by malate dehydrogenase. Produces NADH and a hydrogen ion

Question 73

Net reaction of the citric acid cycle (6 reactants, 6 products)

Answer 73

Reactants: Acetyl CoA, 3 NAD+, FAD, ADP, Pi, 2 water
Products: 2 Co2, 3 NADH, FADH2, ATP, 2 H+, CoA

Question 74

Each pair of electrons from NADH will generate how much ATP?

Answer 74

Each pair of electrons from NADH will generate ~2.5 ATP
when used to reduce oxygen in the electron-transport
chain.

Question 75

Each pair of electrons from FADH2 will generate how much ATP?

Answer 75

Each pair of electrons from FADH2 will power the
synthesis of ~1.5 ATP with the reduction of oxygen in the
electron-transport chain

Question 76

Substrate channeling

Answer 76

The process of the direct transfer of an intermediate between the active sites of two enzymes that catalyze
sequential reactions in a biosynthetic pathway. The close arrangement of enzymes in the citric acid cycle is an example of this- it enhances the efficiency of the cycle because a reaction product can pass directly from one active site to the next through connecting channels

Question 77

Why is the citric acid cycle aerobic?

Answer 77

Molecular oxygen is not directly involved, but the cycle operates only under aerobic conditions because NAD+ and FAD can be regenerated in the mitochondria only by the transfer of electrons to molecular oxygen. NADH and FADH2 are used and their oxidized forms are released. Glycolysis may proceed under anaerobic conditions only because NAD+ is regenerated in the conversion of pyruvate into lactate or ethanol

Question 78

What happens to the carbon atoms that enter the citric acid cycle?

Answer 78

Two carbon atoms enter in the form of an acetyl unit, and
two carbons leave in the form of CO2 molecules (though isotope labeling studies indicate that they are not the same carbon atoms that immediately leave). The two carbon atoms that enter the cycle are retained during the initial two decarboxylation reactions and then remain incorporated in the 4 carbon acids of the cycle. They can occupy any of the carbon positions in the subsequent metabolism of the 4 carbon acids. The two carbons that enter the cycle as the acetyl groups will be released as carbon dioxide in subsequent trips through the cycle

Question 79

How many pairs of the electrons leave the citric acid cycle?

Answer 79

Four pairs of electrons leave on the reduced form of
electron carriers (three NADH and one FADH2).

Question 80

How many NTPs are produced in the citric acid cycle?

Answer 80

One NTP (usually ATP) is generated.

Question 81

How many water molecules are produced by the citric acid cycle?

Answer 81

Two water molecules are consumed: one in the
synthesis of citrate by the hydrolysis of citryl CoA and the
other in the hydration of fumarate.

Question 82

Why must the pyruvate dehydrogenase complex be regulated?

Answer 82

Glucose can be formed from pyruvate, however, the formation of acetyl CoA from pyruvate is an irreversible step in animals, and they are unable to convert acetyl CoA back into glucose. The oxidative decarboxylation of pyruvate commits glucose’s carbon atoms to one of two fates- the citric acid cycle or incorporation into a lipid. The PDC enzyme is therefore located at a critical branch point in metabolism, and it must be heavily regulated

Question 83

2 main fates of acetyl CoA

Answer 83

Metabolism by the
citric acid cycle or incorporation into fatty acids

Question 84

2 regulatory strategies of the pyruvate dehydrogenase complex

Answer 84

Allosteric regulation and reversible phosphorylation

Question 85

Allosteric regulation of the pyruvate dehydrogenase complex

Answer 85

High concentrations of reaction products inhibit the reaction. Acetyl CoA inhibits the transacetylase component (E2) by binding directly. NADH inhibits E3. High concentrations of NADH and acetyl CoA inform the enzyme that the energy needs of the cell have been met, or that fatty acids are being degraded to produce acetyl CoA and NADH. Therefore, it’s not necessary to metabolize pyruvate to make acetyl CoA. This prevents glucose from being metabolized unnecessarily.

Question 86

Covalent modification of the pyruvate dehydrogenase complex

Answer 86

Phosphorylation of the E1 component by pyruvate dehydrogenase kinase switches off the activity of the complex

Question 87

Pyruvate dehydrogenase kinase (PDK)

Answer 87

Switches off the activity of the pyruvate dehydrogenase complex when the E1 component is phosphorylated. There are 4 isozymes of PDK that are expressed in a tissue specific manner

Question 88

Pyruvate dehydrogenase phosphatase (PDP)

Answer 88

Reverses the phosphorylation of the pyruvate dehydrogenase complex (which PDK was responsible for. Once the complex is no longer phosphorylated, it’s active again. There are 2 isozymic forms of the enzyme.

Question 89

How are PDK and PDP regulated?

Answer 89

In muscle at rest for example, the energy demands will not be high. Therefore, the NADH/NAD+, acetyl CoA/CoA, and ATP/ADP ratios will be high. These high ratios promote phosphorylation and inactivation of the complex by activating PDK. This means that high concentrations of immediate (acetyl CoA and NADH) and ultimate (ATP) products inhibit the activity. Therefore, pyruvate dehydrogenase is switched off when the energy charge is high. Conversely, high ADP and pyruvate stimulate the complex.

Question 90

How will pyruvate dehydrogenase complex activity change in active muscle?

Answer 90

As exercise begins, the concentrations of ADP and pyruvate will increase as muscle contraction consumes ATP and glucose is converted into pyruvate to meet energy demands. Both ADP and pyruvate activate the dehydrogenase by inhibiting the kinase. The phosphatase is also stimulated by Ca+, the same signal that initiates muscle contraction

Question 91

Pyruvate dehydrogenase phosphatase deficiency

Answer 91

In people with this deficiency, pyruvate dehydrogenase is always phosphorylated and therefore is inactive. This results in glucose being processed to lactate rather than acetyl CoA. People experience an incessantly high level of lactic acid in the blood. The acidic environment causes many tissues to malfunction, especially the tissues of the central nervous system

Question 92

Primary control points of the citric acid cycle (2)

Answer 92

The reactions catalyzed by the allosteric enzymes isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase. These are the first two enzymes in the cycle to generate high energy electrons

Question 93

Isocitrate dehydrogenase

Answer 93

A control site of the citric acid cycle. The enzyme is allosterically stimulated by ADP, which enhances the enzyme’s affinity for substrates. In contrast, ATP is inhibitory. The reaction product NADH also inhibits the enzyme by directly displacing NAD+- several steps in the cycle require NAD+ or FAD, which are abundant only when the energy charge is low

Question 94

Alpha-ketoglutarate dehydrogenase

Answer 94

Catalyzes the rate limiting step in the citric acid cycle. This enzyme is evolutionarily similar to the pyruvate dehydrogenase complex and some aspects of its control are homologous to the PDC. Alpha-ketoglutarate dehydrogenase is inhibited by succinyl CoA and NADH, the products of the reaction that is catalyzes. The enzyme is also inhibited by a high energy charge. Therefore, the rate of the cycle is reduced when the cell has a high level of ATP.

Question 95

The citric acid cycle is regulated primarily by

Answer 95

The concentration of ATP and NADH

Question 96

4 cellular respiration enzymes that contribute to the development of cancer

Answer 96

1. Succinate dehydrogenase
2. Fumarase
3. Pyruvate dehydrogenase kinase
4. Isocitrate dehydrogenase

Question 97

Aerobic glycolysis (cancer cells)

Answer 97

Cancer cells preferentially metabolize glucose to lactate even in the presence of oxygen. Mutations that alter the activity of succinate dehydrogenase, fumarase, or pyruvate dehydrogenase kinase enhance aerobic glycolysis.

Question 98

Hypoxia inducible factor 1 (HIF-1)

Answer 98

A transcription factor that regulates the enzymes and transporters that enhance glycolysis only when oxygen concentration falls (hypoxia). Normally, HIF-1 is hydroxylated by prolyl hydroxylase 2 and is then destroyed by a complex of proteolytic enzymes. The degradation of HIF-1 prevents the stimulation of glycolysis. Prolyl hydroxylase 2 requires alpha ketoglutarate, vitamin C, and oxygen. Therefore, when oxygen concentration falls, the prolyl hydroxylase 2 is inactive, HIF-1 is not hydroxylated and not degraded, and the synthesis of proteins required for glycolysis is stimulated. This causes glycolysis to increase. Defects in the enzymes of the citric acid cycle may affect the regulation of prolyl hydroxylase 2.

Question 99

How do defects in the citric acid cycle enzymes impact the regulation of prolyl hydroxylase 2?

Answer 99

When succinate dehydrogenase or fumarase is defective, succinate and fumarate accumulate in the mitochondria and spill over into the cytoplasm. Succinate and fumarate act as competitive inhibitors of prolyl hydroxylase 2. When prolyl hydroxylase 2 is inhibited, HIF-1 is not hydroxylated and is stabilized. Lactate (the end product of glycolysis) appears to inhibit prolyl hydroxylase 2 by interfering with the action of vitamin C

Question 100

PDK mutations in cancer

Answer 100

HIF-1 also stimulates the production of PDK. This enzyme inhibits the pyruvate dehydrogenase complex and prevents the conversion of pyruvate into acetyl CoA. The pyruvate remains in the cytoplasm and further increases the rate of aerobic glycolysis. Mutations in PDK that lead to enhanced activity contribute to increased aerobic glycolysis and the subsequent development of cancer. PDK mutations enhance glycolysis and increase the concentration of lactate, resulting in the inhibition of hydroxylase and the stabilization of HIF-1.

Question 101

Isocitrate dehydrogenase and cancer

Answer 101

Mutations in isocitrate dehydrogenase result in the generation of 2-hydroxyglutarate, which is an oncogenic metabolite. The mutant enzyme catalyzes the conversion of isocitrate to alpha-ketoglutarate, but then reduced alpha-ketoglutarate to form 2-hydroxyglutarate. This metabolite alters the methylation patterns in DNA and reduces dependence on growth factors for proliferation. This ultimately alters gene expression and promotes unrestrained cell growth

Question 102

Acetyl CoA acetyltransferase

Answer 102

A mitochondrial enzyme that synthesizes ketone bodies (like acetoacetate) which are a fuel source for tissues. They are an essential fuel source for all tissues under starvation conditions.

Question 103

Acetyl CoA transferase and cancer

Answer 103

In certain cancers, acetyl CoA acetyltransferase is phosphorylated, which causes the enzyme to form active tetramers. The enzyme then acts as protein acetyltransferase, adding acetyl groups to pyruvate dehydrogenase and PDP. This acetylation inhibits these enzymes and facilitates the metabolic switch from oxidative phosphorylation to aerobic glycolysis (enhancing the Warburg effect). The enzyme is hijacked from its normal lipid metabolism function to enhance cancer growth

Question 104

Many of the components of the citric acid cycle are
precursors for

Answer 104

biosynthesis of key biomolecules

Question 105

What other metabolic pathways does the citric acid cycle integrate?

Answer 105

As a major metabolic hub of the cell, the citric acid cycle integrates many of the cell’s other metabolic pathways, including those that involve the synthesis and degradation of carbohydrates, fats, amino
acids, and other important molecules.

Question 106

Why must the citric acid cycle be able to be replenished?

Answer 106

Citric acid cycle intermediates must be replenished if they are used for biosynthetic pathways. If, for example, oxaloacetate is converted into amino acids for protein synthesis and then the energy needs of the cell rise, the citric acid cycle will not be able to operate until new oxaloacetate is formed. Acetyl CoA can’t enter the cell without condensing with oxaloacetate

Question 107

Pyruvate carboxylase

Answer 107

Catalyzes the reaction where oxaloacetate is formed by the carboxylation of pyruvate. Requires carbon dioxide, ATP, and water, and releases ADP and hydrogen ions. This is how oxaloacetate is replenished when used by the cell. Pyruvate carboxylase is a biotin dependent enzyme. Recall that this reaction is also used in gluconeogenesis and is dependent on the presence of acetyl CoA.

Question 108

Anapleurotic reaction

Answer 108

A “filling up” reaction- this describes the reaction catalyzed by pyruvate carboxylase- it
replenishes the supply of a critical citric acid cycle intermediate. The reaction leads to the net synthesis of pathway components

Question 109

Beriberi

Answer 109

A neurological and cardiovascular disorder that is caused by a dietary deficiency of thiamine (vitamin B1). This is a problem in the Far East because rice is a major food and has a low vitamin B1 content. The vitamin thiamine is found in brown rice but not in
white (polished) rice, since thiamine is mostly located in the outer layer of rice

Question 110

Symptoms of beriberi

Answer 110

Neurologic and cardiac symptoms. There is damage to the peripheral nervous system, causing limb pain, muscle weakness, and distorted skin sensation. Patients may have an enlarged heart and inadequate cardiac output

Question 111

How does thiamine deficiency affect biochemical processes?

Answer 111

Thiamine is the precursor of the cofactor thiamine pyrophosphate. This cofactor is the prosthetic group of 3 important enzymes- pyruvate dehydrogenase, alpha ketoglutarate, and transketolase. Its absence prevents the
functioning of those enzymes. The neurologic and cardiovascular disorder beriberi can result.

Question 112

Transketolase

Answer 112

Functions in the pentose phosphate pathway

Question 113

Why does TPP deficiency lead primarily to neurological disorders?

Answer 113

The nervous system relies on glucose as its only fuel. Pyruvate is the product of glycolysis and can enter the citric acid cycle only through the pyruvate dehydrogenase complex. When the PDC is inactive, the nervous system has no other source of fuel. Most other tissues can use fats as a source of fuel

Question 114

Mercury and arsenite poisoning

Answer 114

Both materials have a high affinity for neighboring sulfhydryls, like those in the reduced dihydrolipoyl groups of the E3 component of the PDC. When mercury or arsenite binds to the dihydrolipoyl groups, it inhibits the PDC. This results in CNS pathologies

Question 115

2, 3-Dimercaptopropanol

Answer 115

Can counter the effects of arsenite poisoning by
forming a complex with the arsenite that can be excreted in the urine

Question 116

Where does the phrase “mad as a hatter” originate?

Answer 116

Refers to the strange behavior of poisoned hatmakers who used mercury nitrate to soften and shape animal furs and to make felt. This form of mercury is absorbed through the skin and inhibits the activity of the pyruvate dehydrogenase complex in the brain

Question 117

How did the citric acid cycle come into existence?

Answer 117

The citric acid cycle was most likely assembled from pre-existing reaction pathways. Many of the intermediates formed in the citric acid cycle are used in metabolic pathways for amino acids and porphyrins. Therefore, compounds like pyruvate, alpha ketoglutarate, and oxaloacetate were likely present early in evolution for biosynthetic purposes.

Question 118

Which reactions likely formed the core processes that proceeded the citric acid cycle?

Answer 118

The oxidative decarboxylation of alpha-ketoacids is thermodynamically favorable and can be used to drive the synthesis of both acyl CoA derivatives and NADH. These reactions likely formed the core of processes that preceded the citric acid cycle evolutionarily.

Question 119

Glyoxylate cycle

Answer 119

Similar to the citric acid cycle but
bypasses the two decarboxylation steps, allowing the synthesis of carbohydrates from fats. Acetyl CoA generated from fat stores is converted into glucose. This pathway is present in plants and in some microorganisms, because some organisms can’t convert acetyl CoA into glucose. It allows some microorganisms to grow on acetate. Succinate can be converted into oxaloacetate and then into glucose.

Question 120

Differences between the citric acid cycle and the glyoxylate cycle (2)

Answer 120

1. Two molecules of acetyl CoA enter per turn of the glyoxylate cycle, only one enters for the CAC
2. The glyoxylate cycle skips the 2 decarboxylation steps of the citric acid cycle

Question 121

Isocitrate lyase

Answer 121

Cleaves isocitrate into succinate and glyoxylate in the glyoxylate cycle

Question 122

Malate synthase

Answer 122

Acetyl CoA condenses with glyoxylate to form malate in the glyoxylate cycle. Malate goes on to be oxidized to oxaloacetate, like in the citric acid cycle

Question 123

Sum of the reactions in the glyoxylate cycle (3 reactants, 4 products)

Answer 123

Reactants- 2 acetyl CoA, NAD, and 2 water molecules
Products- succinate, 2 CoA, NADH, and 2 H+

Question 124

Glyoxysomes

Answer 124

Organelles where the glyoxylate cycle occurs in plants. The cycle is prominent in oil-rich seeds, like those from sunflowers and cucumbers.

Question 125

Benefits of the glyoxylate cycle

Answer 125

Succinate is released in the middle of the cycle and can be converted into carbohydrates by a combination of the citric acid cycle and gluconeogenesis. The carbohydrates power the growth of seedlings until the cell can begin photosynthesis. This means that organisms with the glyoxylate cycle can gain metabolic versatility because they can use acetyl CoA as a precursor of glucose and other biomolecules

Question 126

Diabetic neuropathy

Answer 126

A numbness, tingling, or
pain in the limbs and digits, is a common complication of both type 1 and type 2 diabetes. The symptoms may be caused by overproduction of lactic acid by cells in the dorsal root ganglion, a part of the nervous system responsible for pain
perception. It may result from inhibition of the pyruvate dehydrogenase complex

Question 127

Why do diabetics produce more lactic acid?

Answer 127

The increase in lactic acid in diabetics may be due to
hyperglycemia (high glucose), the defining feature of
diabetes. This increases pyruvate dehydrogenase kinase activity
in the cells of the dorsal root ganglion. This kinase then
phosphorylates and inhibits the pyruvate dehydrogenase
complex. Glycolytically produced pyruvate is then converted to
lactate, and excess lactate leads to an increase in acid-sensing pain receptors. This ultimately results in the symptoms of diabetic neuropathy

Question 128

Mycobacterium tuberculosis

Answer 128

The bacterium responsible for tuberculosis (TB)- it is transmitted by people with active lung infections by coughing and sneezing. A common treatment for TB is the antibiotic rifampicin, which acts as an inhibitor of bacterial protein translation. However, as resistant bacterial strains emerge, new treatments are needed

Question 129

Role of the glyoxylate cycle in TB

Answer 129

The bacteria are dependent on the glyoxylate cycle (which allows conversion of fats to glucose), especially when they are in a latent state in the lungs. A key enzyme in the glyoxylate cycle is isocitrate lyase, and a suicide (or mechanism-based)
inhibitor for the lyase has been
synthesized to more effectively treat TB

Question 130

2-vinyl-isocitrate

Answer 130

A mechanism-based lyase inhibitor used to treat TB. In the process of catalysis, a reactive thiolate is formed on cysteine 191 in the active site. When this inhibitor reacts with the lyase, succinate is released as in the normal reaction. However, the cysteine of the lyase remains
covalently modified. Because this cysteine is conserved
in all M. tuberculosis strains, the
likelihood of evolving resistance to the drug is diminished.