Chapter 16: The Citric Acid Cycle

Question 1

Cellular respiration is a metabolic pathway that breaks down ______ and produces _____. The stages of cellular respiration include

Answer 1

• glucose
• ATP
• glycolysis, pyruvate oxidation, the citric acid or Krebs cycle, and oxidative phosphorylation.
• PDF pg. 678 & 680

Question 2

glycolysis

prior to cellular respiration

Answer 2

• Means sugar splitting
• Occurs in cytosol
• Breaks glucose into 2 pyruvates
• Releases less than 25% of energy in glucose; most energy remains in pyruvate
• Has 2 phases
  
  • Energy Investment Phase
    
    • Glucose enters cell: (mammals) via facilitated diffusion by GLUT1
    • No oxygen required! Anaerobic
    • Glucose (6-Carbon sugar) is split into two 3-Carbon sugars, G3P (Glyceraldehyde 3-phosphate)
    • Cell spends 2 ATP to perform this conversion
    • Only step that is endergonic (because it needs 2 ATP)
    • ΔG is positive (not spontaneous)
    • 0 CO₂, -2 ATP, 0 NADH, 0 FADH₂
  • Energy Payoff Phase
    
    • two G3P converted to two Pyruvate
    • four ATP are produced by substrate-level phospholyration
    • two NAD⁺ are is reduced to NADH
    • Exergonic
    • ΔG is negative (not spontaneous)
    • 0 CO2, 4 ATP, 2 NADH, 0 FADH2
• 0 CO2, 2 ATP, 2 NADH, 0 FADH2

Question 3

Cellular respiration

first step: pyruvate oxidation

Answer 3

• Two Pyruvate enters mitochondrial matrix
• Each pyruvate is converted to the compound acetyl CoA and CO₂ by pyruvate dehydrogenase complex (PDH) a multienzyme complex
• ​The irreversible reaction catalyzed by PDH is an oxidative decarboxylation
  
  1. Pyruvate’s carboxyl group is removed as a molecule of CO₂
  2. Remaining fragment is oxidized to form acetic acid, the acetyl group of acetyl-CoA
  3. acetic acid is converted to acetyl CoA
  4. e^- removed from step 3 are transferred to NAD⁺ to make NADH
• NADH gives up a hydride ion (:H2) to the respiratory chain, which is then carried to the final electron acceptor (oxygen or in anaerobic organisms to nitrate or sulfate)
• The transfer of electrons from NADH to oxygen ultimately generates 2.5 molecules of ATP per pair of electrons

Question 4

Cellular respiration

first step: pyruvate oxidation:

pyruvate dehydrogenase (PDH)

Answer 4

• in mitochondria of eukaryotic cells and in cytosol of bacteria
• multienzyme complex
• requires the sequential action of three different enzymes
  
  • has multiple copies of the three enzyme
  • conserved during evolution
• five different coenzymes or prosthetic groups (cofactors) remain bound to the enzyme molecules as substrate is transformed
  
  • thiamine pyrophosphate (TPP)
  • flavin adenine dinucleotide (FAD): riboflavin
    
    • electron carrier
  • coenzyme A (CoA, sometimes denoted CoA-SH, to emphasize the role of the —SH group): pantothenate
    
    • ​acyl carrier
  • nicotinamide adenine dinucleotide (NAD): niacin
    
    • ​electron carrier
  • lipoate
    
    • electron/acyl carrier
  • four derived from vitamins
• Also part of the complex are two regulatory proteins, a protein kinase and a phosphoprotein phosphatase
• the prototype for two other important enzyme complexes: ketoglutarate dehydrogenase, of the citric acid cycle

Question 5

Cellular respiration

first step: pyruvate oxidation:

Coenzyme A

Answer 5

• has a reactive thiol —SH group critical to the role of acyl carrier
• Acyl groups are covalently linked to the thiol group, forming thioesters
• Because of their relatively high standard free energies of hydrolysis, —SH have a high acyl group transfer potential, and the acyl group attached is considered “activated”
• structure
  
  • right to left in picture
  • A hydroxyl group of pantothenic acid is joined to a modified ADP moiety by a phosphate ester bond
  • the pantothenic acid carboxyl group is attached to b-mercaptoethylamine in amide linkage
  • hydroxyl group at the 3’ position of the ADP moiety has a phosphoryl group not present in free ADP
  • —SH group of the mercaptoethylamine moiety forms a thioester with acetate in acetyl-coenzyme A (acetyl-CoA)

Question 6

Cellular respiration

first step: pyruvate oxidation:

lipoate

Answer 6

• has two thiol groups that can undergo reversible oxidation to a disulfide bond (—S—S—)
• can serve both as an electron carrier and as an acyl carrier, as we shall see

Question 7

Cellular respiration

first step: pyruvate oxidation:

pyruvate dehydrogenase (PDH)

3 enzymes

Answer 7

• pyruvate dehydrogenase (E₁)
  
  • active site has bound TPP
• dihydrolipoyl transacetylase (E₂)
  
  • the point of connection for the prosthetic group lipoate
  • attached through an amide bond to the ε-amino group of a Lys residue
  • has three functionally distinct domains
    
    • amino-terminal lipoyl domain
      
      • contains the lipoyl-Lys residue(s)
    • central E1- and E3-binding domain
    • innercore acyltransferase domain
  • domains are separated by linkers
    
    • sequences of 20 to 30 amino acid residues
    • rich in Ala and Pro
    • interspersed with charged residues
    • tend to assume extended forms, holding domains apart
• dihydrolipoyl dehydrogenas (E₃)
  
  • active site has bound FAD
• attachment of lipoate to the end of a Lys side chain in E₂ produces a long, flexible arm that can move from the active site of E₁ to the active sites of E₂ and E₃, a distance of perhaps 5 nm or more
  
  • Central to the mechanism of the PDH complex
  • swinging lipoyllysyl arms of E₂, accept two e^- and the acetyl group derived from pyruvate from E₁. E₂ then passes them to E3

Question 8

substrate channeling

Answer 8

• the passing of the intermediary metabolic product of one enzyme directly to another enzyme or active site without its release into solution
• enzymes and coenzymes are usually clustered
• allows intermediates to react quickly without diffusing away from the surface of the enzyme complex
• prevents theft of the activated acetyl group by other enzymes that
• When several consecutive enzymes of a metabolic pathway channel substrates between themselves, this is called a metabolon.

Question 9

Cellular respiration

second step: Citric Acid Cycle

aka Trycarboxylic Acid Cycle and Krebs cycle

Answer 9

• Two Acetyl CoA enters mitochondrial matrix, in prokaryotes this happens in the cytosol and the plasma membrane plays a role analogous to that of the inner mitochondrial membrane
• Acetyl CoA enters the citric acid cyle
• One turn of the cycle per acetyl coA
• Has 8 steps, each catalyzed by a specific enzyme
  
  • Acetyl coA donates its acetyl group to oxaloacetate → citrate
  • Citrate → isocitrate
  • Isocitrate is oxidized → α-Ketoglutarate (aka oxoglutarate) & CO₂
    
    • its electrons reduce NAD⁺ to NADH
    • loses a CO₂
  • Ketoglutarate is oxidized → Succinyl CoA
    
    • its electrons reduce NAD⁺ to NADH
    • loses a CO₂
  • Succinyl CoA → Succinate
    
    • Creating ATP
  • Succinate oxidized → Fumarate
    
    • its electrons reduce FAD to FADH₂
  • Fumarate → Malate
  • Malate oxidized → Oxaloacetate
    
    • its electrons reduce NAD to NADH₂
    • Oxaloacetate ready to react with another Acetyl coA
• Dehydrogenases transfer electrons to NADH
• Four and five-carbon intermediates serve as precursors for other products; cells employ anaplerotic (replenishing) reactions
• PDF pg. 669
• Note: the two carbon atoms in CO₂ released are not the same two carbons that entered in the form of the acetyl group; additional turns around the cycle are required to release these carbons as CO₂

Question 10

Cellular respiration

second step: Citric Acid Cycle

aka Trycarboxylic Acid Cycle and Krebs cycle

step 1 of 8: formation of citrate

Answer 10

• condensation of Acetyl coA with oxaloacetate → citrate
  
  • Acetyl coA donates its acetyl group to oxaloacetate
• catalyzed by citrate synthase a Claisen condensation reaction
  
  • homodimeric enzyme
  • single polypeptide with two domains, one large and rigid, the other smaller and more flexible
  • active site between domains
  • Induced fit to its substrate and intermediate decreases premature and unproductive cleavage
• Reaction steps
  
  • Oxaloacetate binds first inducing a large conformational change in the flexible domain
  • this creates a binding site for Acetyl coA
  • methyl carbon of the acetyl group is joined to the carbonyl group (C-2) of oxaloacetate
  • formation of transient intermediate Citroyl-CoA causes another conformational change and hydrolysis (highly exogernic) to free CoA and citrate
  • liberated CoA is recycled in the oxidative decarboxylation of another pyruvate by the PDH complex
• PDF pg. 671

Question 11

Cellular respiration

second step: Citric Acid Cycle

aka Trycarboxylic Acid Cycle and Krebs cycle

step 3 of 8: formation of citrate

Answer 11

• Citrate → isocitrate
• aconitase (aconitate hydratase) catalyzes the reversible transformation of citrate to isocitrate
  
  • forms the intermediate tricarboxylic acid cis-aconitate
  • can promote the reversible addition of H₂O to the double bond cis-aconitate in two different ways, one leading to citrate and the other to isocitrate
  • reaction is pulled to the right because isocitrate is rapidly consumed in the next step
• Aconitase contains an ironsulfur center
  
  • acts in the binding of the substrate at the active site and in the catalytic addition or removal of H2O
  • In iron-depleted cells, it loses its iron-sulfur center and acquires the ability to bind to mRNA for the transferring receptor/ferritin, regulating protein synthesis at the translational level & iron homeostasis
  • PDF pg. 673
• PDF pg. 672

Question 12

Cellular respiration

second step: Citric Acid Cycle

aka Trycarboxylic Acid Cycle and Krebs cycle

step 3 of 8: Isocitrate is oxidized → α-Ketoglutarate and CO₂

Answer 12

• Isocitrate is oxidized → Ketoglutarate (aka oxoglutarate)
  
  • its electrons reduce NAD+ to NADH
  • loses a CO2
• isocitrate dehydrogenase catalyzes oxidative decarboxylation of isocitrate
  
  • two types
    
    • one requiring NAD^+​
      
      • In the mitochondria
      • serves in the citric acid cycle
    • one requiring NADP⁺
      
      • ^​In the mitochondria and cytosol
      • generation of NADPH, essential for reductive anabolic reactions
    • reactions are identical
• Reaction steps
  
  • Mn²⁺ in the active site interacts with the carbonyl group of the enol intermediate oxalosuccinate, and stabilizes it
  • Rearrangement of the enol intermediate generates α-Ketoglutarate
• PDF pg. 674

Question 13

Cellular respiration

second step: Citric Acid Cycle

aka Trycarboxylic Acid Cycle and Krebs cycle

step 4 of 8: α-Ketoglutarate is oxidized → Succinyl CoA and CO₂

Answer 13

• α-Ketoglutarate is oxidized → Succinyl CoA and CO₂
  
  • its electrons reduce NAD+ to NADH
  • loses a CO₂
• α-Ketoglutarate dehydrogenase complex catalyzes oxidative decarboxylation of α-Ketoglutarate
  
  • closely resembles the PDH complex in both structure and function
  • three enzymes, homologous to E₁, E₂, and E₃ of the PDH complex, as well as enzyme-bound TPP, bound lipoate, FAD, NAD, and coenzyme A
  • E₁ are structurally similar but their amino acid sequences differ
    
    • PDH complex binds pyruvate
    • α-Ketoglutarate binds α-ketoglutarate
  • E₂ are very similar
  • E₃ are identical
  • case of divergent evolution: genes for an enzyme with one substrate specificity give rise, during evolution, to closely related enzymes with different substrate specificities but the same enzymatic mechanism
• The energy of oxidation of α-ketoglutarate is conserved in the formation of the thioester bond of succinyl-CoA

Question 14

Cellular respiration

second step: Citric Acid Cycle

aka Trycarboxylic Acid Cycle and Krebs cycle

step 5 of 8: Succinyl CoA → Succinate

Answer 14

• Succinyl CoA → Succinate
  
  • Creating ATP
• Succinyl-CoA, like acetyl-CoA, has a thioester bond with a strongly
  negative standard free energy of hydrolysis (ΔG’° ≈ -36 kJ/mol)
  
  • energy released in breakage of this bond drives the synthesis of a phosphoanhydride bond in GTP or ATP with a net ΔG’° of only -2.9 kJ/mol
• succinyl-CoA synthetase (succinic thiokinase) catalyzes the reversible reaction
  
  • Animal cells have two isozymes of succinyl-CoA synthetase
    
    • one specific for ADP
    • the other for GDP
    • net result of either isozyme is the conservation of energy as ATP
  • has two subunits
    
    • α (M_r 32,000) has the ℗–His residue (His²⁴⁶) and the binding site for CoA
    • β (M_r 42,000) confers specificity for either ADP or GDP
  • The active site is at the interface between subunits
  • has two “power helices”
    
    • one from each subunit
    • oriented so their electric dipoles situate partial positive charges close to the negatively charged ℗–His
    • stabilizes the phosphoenzyme intermediate
• Reaction steps
  
  • energy-conserving reaction
  • succinyl-CoA binds to the enzyme
  • a phosphoryl group replaces the CoA of succinyl-CoA, forming a high-energy acyl phosphate
  • the succinyl phosphate donates its phosphoryl group to a His residue in the active site of the enzyme, forming a high-energy phosphohistidyl enzyme
  • the phosphoryl group is transferred from the His residue to the terminal phosphate of GDP (or ADP), forming GTP (or ATP)
    
    • formation of ATP (or GTP) at the expense of the energy released by the oxidative decarboxylation of α-ketoglutarate is a substrate-level phosphorylation
  • net result of the
• GTP formed by succinyl-CoA synthetase can donate its terminal phosphoryl group to ADP to form ATP
  
  • reversible reaction catalyzed by nucleoside diphosphate kinase
  • net result is conservation of energy in ATP
  • No change in free energy for reaction; ATP and GTP are energetically equivalent

Question 15

Cellular respiration

second step: Citric Acid Cycle

aka Trycarboxylic Acid Cycle and Krebs cycle

step 6 of 8: Succinate oxidized → Fumarate

Answer 15

• Succinate oxidized → Fumarate
  
  • its electrons reduce FAD to FADH2
• flavoprotein succinate dehydrogenase oxidizes Succinate
  
  • In eukaryotes it’s in mitochondrial inner membrane
  • in bacteria in plasma membrane
  • contains three different iron-sulfur clusters and one molecule of covalently bound FAD
  • e^- pass from succinate through the FAD and iron-sulfur centers before entering the chain of e^- carriers in the membrane, to the final e^- acceptor
  • synthesizes about 1.5 ATP per pair of e^-
• Malonate
  
  • analog of succinate
  • not normally present in cells
  • strong competitive inhibitor of succinate dehydrogenase
  • addition to mitochondria blocks activity of the citric acid cycle

Question 16

Cellular respiration

second step: Citric Acid Cycle

aka Trycarboxylic Acid Cycle and Krebs cycle

step 7 of 8: Fumarate → Malate

Answer 16

• Reversrible Hydration of Fumarate → Malate
• catalyzed by fumarase
• transition state is a carbanion
• enzyme is highly stereospecific
  
  • it catalyzes hydration of the trans double bond of fumarate but not the cis double bond of maleate
  • In the reverse direction (from L-malate to fumarate), fumarase is equally stereospecific: D-malate is not a substrate
• PDF pg. 678

Question 17

Cellular respiration

second step: Citric Acid Cycle

aka Trycarboxylic Acid Cycle and Krebs cycle

step 8 of 8: Malate oxidized → Oxaloacetate

Answer 17

• Malate oxidized → Oxaloacetate
  
  • its electrons reduce NAD to NADH₂
  • Oxaloacetate ready to react with another Acetyl coA
• L-malate dehydrogenase catalyzes the oxidation
• PDF pg. 678

Question 18

Synthetases

Answer 18

catalyze condensations that use ATP or another nucleoside triphosphate as a source of energy

Question 19

Synthases

Answer 19

catalyze condensation reactions in which no nucleoside triphosphate (ATP, GTP, and so forth) is required as an energy source

Question 20

Ligases

Answer 20

• catalyze condensation reactions in which two atoms are joined, using ATP or another energy source
• Thus synthetases are ligases

Question 21

lyases

Answer 21

catalyze cleavages (or, in the reverse direction, additions) in which electronic rearrangements occur

Question 22

kinase

Answer 22

• enzymes that transfer a phosphoryl group from a nucleoside triphosphate such as ATP to an acceptor molecule—a sugar, protein, another nucleotide, or a metabolic intermediate
• Reaction catalyzed by a kinase is a phosphorylation

Question 23

phosphorylases

Answer 23

• phosphorolysis
• a displacement reaction in which phosphate is the attacking species and becomes covalently attached at the point of bond breakage

Question 24

phosphatases

Answer 24

• Dephosphorylation
• removal of a phosphoryl group from a phosphate with water as the attacking species

Question 25

Cellular respiration

second step: Citric Acid Cycle

Products of one turn of the citric acid cycle during roxidative decarboxylation reactions

Answer 25

• 3 NADH
• 1 FADH₂
• 1 GTP (or ATP)
• 2 CO₂ are released in oxidative decarboxylation reactions.
• PDF pg. 680

Question 26

Under some metabolic circumstances, intermediates are drawn out of the cycle to be used as ______ in a variety of biosynthetic ______. some modern anaerobic microorganisms use an incomplete citric acid cycle as a source of biosynthetic ______.

Answer 26

• precursors
• pathways
• precursors

Question 27

• In aerobic organisms, the citric acid cycle is an ______ ______, one that serves in both catabolic and anabolic processes.
• Besides its role in the oxidative catabolism of carbohydrates, fatty acids, and amino acids, the cycle provides precursors for many biosynthetic pathways such as:

Answer 27

• amphibolic pathway
• precursors:
  
  • α-Ketoglutarate and oxaloacetate serve as precursors of the amino acids aspartate and glutamate which are then used to build other amino acids, as well as purine and pyrimidine nucleotides
  • Succinyl-CoA is a central intermediate in the synthesis of the porphyrin ring of heme groups

Question 28

As intermediates of the citric acid cycle are removed to serve as biosynthetic precursors, they are replenished by ______ _____, so that the concentrations of the citric acid cycle intermediates remain almost constant.

Answer 28

anaplerotic reactions

Question 29

• The most important anaplerotic reaction in mammalian liver and kidney is the reversible carboxylation of pyruvate by CO₂ to form oxaloacetate, catalyzed by ______ _____.
• When the citric acid cycle is deficient in oxaloacetate or any other intermediates, pyruvate is carboxylated to produce more oxaloacetate which requires ______.
• Pyruvate carboxylase is a ______ enzyme and is virtually inactive in the absence of ______, its positive allosteric modulator.
• Whenever _______, the fuel for the citric acid cycle, is present in excess, it stimulates the pyruvate carboxylase reaction to produce more ______, enabling the cycle to use more acetylCoA in the citrate synthase reaction

Answer 29

• pyruvate carboxylase
• ATP
• regulatory, acetyl-CoA
• acetyl-CoA, oxaloacetate
• PDF pg. 681

Question 30

• The pyruvate carboxylase reaction requires the vitamin _____, which is the prosthetic group of the enzyme
• It is a specialized carrier of one-carbon groups in their most oxidized form:
• Carboxyl groups are activated in a reaction that consumes ATP and joins CO₂ to enzyme-bound biotin. This “activated” CO₂ is then passed to an ______ (pyruvate in this case) in a _____ ______
• protein ______ (Mr 70,000) binds very tightly to biotin and prevents its absorption in the intestine

Answer 30

• biotin
• CO₂
• acceptor
• carboxylation reaction
• avidin

Question 31

Pyruvate carboxylase structure

Answer 31

• has four identical subunits
• each containing a molecule of biotin covalently attached through an amide linkage to the ε-amino group of a specific Lys residue in the enzyme active site
• Carboxylation of pyruvate proceeds in two steps, in separate active sites
  
  • first: carboxyl group derived from HCO₃^- is attached to biotin
  • second: carboxyl group is transferred to pyruvate to form oxaloacetate
  • the long flexible arm of biotin transfers activated carboxyl groups from the first active site (on one monomer of the tetramer) to the second (on the adjacent monomer)
• PDF pg 683

Question 32

______, ______, and ______ all enter cells on the same transporter, become covalently attached to proteins by similar reactions, and provide a flexible tether that allows bound reaction intermediates to move from one active site to another in an enzyme complex, without dissociating from it, participating in ______ ______.

Answer 32

• Lipoate, biotin, and pantothenate
• substrate channeling

Question 33

Regulation of the Citric Acid Cycle

Answer 33

• The flow of carbon atoms from pyruvate into and through the citric acid cycle is under tight regulation at two levels:
  
  • the conversion of pyruvate to acetyl-CoA (pyruvate dehydrogenase complex reaction)
  • the entry of acetyl-CoA into the cycle (the citrate synthase reaction)
• cycle is also regulated at the isocitrate dehydrogenase and α-ketoglutarate dehydrogenase reactions.

Question 34

PDH complex inhibition

Answer 34

• strongly inhibited by products of the reaction: ATP, acetyl-CoA and NADH. activity is turned off when
  
  • when the cell’s [ATP]/[ADP] and [NADH]/[NAD1] ratios are high
  • ample fuel is available in the form of fatty acids and acetyl-CoA
• allosteric activated by AMP, CoA, and NAD1, which accumulate when too little acetate flows into the citric acid cycle. activity is turned on
  
  • when energy demands are high and the cell requires greater flux of acetyl-CoA into the citric acid cycle
• in mammals, complemented by a second level of regulation: covalent protein modification.
  
  • inhibited by reversible phosphorylation of a specific Ser residue on one of the two subunits of E1
  • Pyruvate dehydrogenase kinase phosphorylates and thereby inactivates E1, and a specific phosphoprotein phosphatase removes the phosphoryl group by hydrolysis and thereby activates E1
  • When [ATP] declines, kinase activity decreases and phosphatase action removes the phosphoryl groups from E1, activating the complex
• PDF pg. 685

Question 35

Three factors govern the rate of flux through the citric acid cycle:

Answer 35

• substrate availability
• inhibition by accumulating products
• allosteric feedback inhibition

Question 36

• the three strongly exergonic steps in the cycle catalyzed by _____ _____, _____ _____, and _____ _____ can become the rate-limiting step under some circumstances
• availability of the substrates for citrate synthase, _____ and _____ varies, limiting the rate of citrate formation
• _____, a product of isocitrate and α-ketoglutarate oxidation, accumulates under some conditions, and at high ______ both dehydrogenase reactions are severely inhibited
• malate dehydrogenase reaction is at equilibrium but when _____ is high the concentration of _____ is low, slowing the first step in the cycle
• _____ _____ inhibits all three limiting steps

Answer 36

• citrate synthase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase
• acetyl-CoA, oxaloacetate
• NADH, [NADH]/[NAD1]
• [NADH]/[NAD1], oxaloacetate
• Product accumulation
• PDF pf 686

Question 37

metabolon

Answer 37

• transient multi-protein complexes of sequential enzymes that mediate substrate channeling
• They differ from multi-enzyme complexes in that they are dynamic, rather than permanent, and as such have considerably lower dissociation constants
• formed between sequential enzymes of a metabolic pathway, held together both by non-covalent interactions and by structural elements of the cell, such as integral membrane proteins and proteins of the cytoskeleton

Question 38

The Glyoxylate Cycle

Answer 38

• In plants, certain invertebrates, and some microorganisms acetate can serve as an energy-rich fuel and as a source of phosphoenolpyruvate for carbohydrate synthesis
• glyoxylate cycle catalyze the net conversion of acetate to succinate or other four-carbon intermediates of the citric acid cycle
• steps
  
  • acetyl-CoA condenses w/oxaloacetate to form citrate
  • citrate is converted to isocitrate, like in the citric acid cycle
  • isocitrate is cleved by isocitrate lyase, forming succinate and glyoxylate
  • glyoxylate condenses with 2nd molecule of acetyl-CoA to yield malate, in a reaction catalyzed by malate synthase
  • malate is oxidized to oxaloacetate, which can condense with another molecule of acetyl-CoA to start another turn of the cycle
• Each turn of the glyoxylate cycle
  
  • consumes two molecules of acetyl-CoA
  • produces one molecule of succinate, which is then available for biosynthetic purposes
• PDF pg 688