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.

Chapter 16: The Citric Acid Cycle Flashcards by Alexandra Miranda

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

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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
0 CO2, 2 ATP, 2 NADH, 0 FADH2

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Cellular respiration

first step: pyruvate oxidation

Two Pyruvate enters mitochondrial matrix
Each pyruvate is converted to the compound acetyl CoA and CO₂by 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

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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

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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)

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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

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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
- 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. E₂then passes them to E3

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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.

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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

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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

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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

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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
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

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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

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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 (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

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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

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Cellular respiration

second step: Citric Acid Cycle

aka Trycarboxylic Acid Cycle and Krebs cycle

step 7 of 8: Fumarate → Malate

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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

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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

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

Synthases

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

Ligases

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

lyases

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

kinase

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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

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

phosphatases

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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

Chapter 16: The Citric Acid Cycle Flashcards

(38 cards)