Chapter 16: The Citric Acid Cycle Flashcards
Cellular respiration is a metabolic pathway that breaks down ______ and produces _____. The stages of cellular respiration include
- glucose
- ATP
- glycolysis, pyruvate oxidation, the citric acid or Krebs cycle, and oxidative phosphorylation.
- PDF pg. 678 & 680

glycolysis
prior to cellular respiration
- 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
- Energy Investment Phase
- 0 CO2, 2 ATP, 2 NADH, 0 FADH2

Cellular respiration
first step: pyruvate oxidation
- 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
- Pyruvate’s carboxyl group is removed as a molecule of CO2
- Remaining fragment is oxidized to form acetic acid, the acetyl group of acetyl-CoA
- acetic acid is converted to acetyl CoA
- 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

Cellular respiration
first step: pyruvate oxidation:
pyruvate dehydrogenase (PDH)
- 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

Cellular respiration
first step: pyruvate oxidation:
Coenzyme A
- 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)

Cellular respiration
first step: pyruvate oxidation:
lipoate
- 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
Cellular respiration
first step: pyruvate oxidation:
pyruvate dehydrogenase (PDH)
3 enzymes
- 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
- amino-terminal lipoyl 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

substrate channeling
- 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.
Cellular respiration
second step: Citric Acid Cycle
aka Trycarboxylic Acid Cycle and Krebs cycle
- 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
Cellular respiration
second step: Citric Acid Cycle
aka Trycarboxylic Acid Cycle and Krebs cycle
step 1 of 8: formation of citrate
- 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

Cellular respiration
second step: Citric Acid Cycle
aka Trycarboxylic Acid Cycle and Krebs cycle
step 3 of 8: formation of citrate
- 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
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
- 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
- one requiring NAD+
- two types
- 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
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
- 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

Cellular respiration
second step: Citric Acid Cycle
aka Trycarboxylic Acid Cycle and Krebs cycle
step 5 of 8: Succinyl CoA → Succinate
- 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 (Mr 32,000) has the ℗–His residue (His246) and the binding site for CoA
- β (Mr 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
- Animal cells have two isozymes of succinyl-CoA synthetase
- 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

Cellular respiration
second step: Citric Acid Cycle
aka Trycarboxylic Acid Cycle and Krebs cycle
step 6 of 8: Succinate oxidized → Fumarate
- 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
Cellular respiration
second step: Citric Acid Cycle
aka Trycarboxylic Acid Cycle and Krebs cycle
step 7 of 8: Fumarate → Malate
- 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

Cellular respiration
second step: Citric Acid Cycle
aka Trycarboxylic Acid Cycle and Krebs cycle
step 8 of 8: Malate oxidized → Oxaloacetate
- 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

Synthetases
catalyze condensations that use ATP or another nucleoside triphosphate as a source of energy
Synthases
catalyze condensation reactions in which no nucleoside triphosphate (ATP, GTP, and so forth) is required as an energy source
Ligases
- catalyze condensation reactions in which two atoms are joined, using ATP or another energy source
- Thus synthetases are ligases
lyases
catalyze cleavages (or, in the reverse direction, additions) in which electronic rearrangements occur
kinase
- 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
phosphorylases
- phosphorolysis
- a displacement reaction in which phosphate is the attacking species and becomes covalently attached at the point of bond breakage
phosphatases
- Dephosphorylation
- removal of a phosphoryl group from a phosphate with water as the attacking species
Cellular respiration
second step: Citric Acid Cycle
Products of one turn of the citric acid cycle during roxidative decarboxylation reactions
- 3 NADH
- 1 FADH2
- 1 GTP (or ATP)
- 2 CO2 are released in oxidative decarboxylation reactions.
- PDF pg. 680

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 ______.
- precursors
- pathways
- precursors
- 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:
- 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
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.
anaplerotic reactions
- 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
- pyruvate carboxylase
- ATP
- regulatory, acetyl-CoA
- acetyl-CoA, oxaloacetate
- PDF pg. 681
- 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
- biotin
- CO2
- acceptor
- carboxylation reaction
- avidin
Pyruvate carboxylase structure
- 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
______, ______, 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 ______ ______.
- Lipoate, biotin, and pantothenate
- substrate channeling
Regulation of the Citric Acid Cycle
- 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.
PDH complex inhibition
- 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

Three factors govern the rate of flux through the citric acid cycle:
- substrate availability
- inhibition by accumulating products
- allosteric feedback inhibition
- 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
- citrate synthase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase
- acetyl-CoA, oxaloacetate
- NADH, [NADH]/[NAD1]
- [NADH]/[NAD1], oxaloacetate
- Product accumulation
- PDF pf 686
metabolon
- 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
The Glyoxylate Cycle
- 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