Chapter 17- Pyruvate Dehydrogenase and the citric acid cycle

Citric Acid Cycle

series of oxidation–reduction reactions that result in the oxidation of an acetyl group to two molecules of CO₂

• the final pathway for the oxidation of fuel molecules

• oxidation generates high-energy electrons used to power ATP synthesis

• important sources of precursors for biosynthesis

• also called the tricarboxylic acid (TCA) cycle or Krebs cycle

Acetyl CoA

Most fuel molecules enter the citric acid cycle as acetyl CoA (acetyl coenzyme A)

Pyruvate dehydrogenase complex

A large enzyme complex that oxidatively decarboxylates pyruvate to acetyl CoA under aerobic conditions

Acetyl CoA’s entrance into the citric acid cycle

Acetyl CoA enters the citric acid cycle where all remaining carbons are completely oxidized to CO₂

Reactions to the pyruvate dehydrogenase complex and the citric acid cycle take place in the mitochondrial matrix

Structure of the mitochondria

• Have distinct compartments defined by two membranes

• In eukaryotes, the reactions of the citric acid cycle take place in the matrix of the mitochondria

• Glycolysis takes place in the cytoplasm

Overview of the citric acid cycle

The citric acid cycle removes electrons from acetyl CoA and uses the electrons to reduce NAD⁺ and FAD to form NADH and FADH₂

Electron-transport Chain

A series of membrane proteins that electrons released in the rexoidation of NADH and FADH₂ flow through to generate a proton gradient across the inner mitochondrial membrane

• Protons flow through ATP synthase to generate ATP from ADP and inorganic phosphate

How does citric acid connect to the elctron transport chain?

Cellular respiration removes high-energy electrons from carbon fuel molecules to generate ATP

How does the citric acid cycle connect to the electron transport chain?

Through pyruvate dehydrogenase

• Pyruvate dehydrogenase complex: a highly integrated unit of three distinct enzymes in the mitochondrial matrix

• oxidatively decarboxylates pyruvate to acetyl CoA

  • Pyruvate + CoA + NAD⁺ → acetyl CoA + CO₂ + NADH + H⁺

• Reaction catalyzed by the pyruvate dehydrogenase coomplex is an irreverisble link between glycolysis and the citric acid cycle

Conversion of pyruvate into acetyl CoA

Consists of three steps plus a regeneration steo

• steps must be coupled because the free energy from the decarboxylation step drives the formation of NADH and acetyl CoA

• The catalytic cofactors are thiamine pyrophosphate (TPP), lipoic acid, and FAD

• The stoichiometrix cofactors (cofactors that function as substrates) are CoA and NAD⁺

Citric Acid cycle step 1

Citric Acid cycle oxidizes two-carbon units

Citric synthase catalyzes the addition of acetyl CoA and oxaloacetate, yielding citrate and CoA

• reaction is an aldol addition and a hydrolysis

• proceeds through energy-rich citryl CoA

Citric Acid cycle step 2

Mechanism of citrate synthease prevents undesirable reactions

• it minimizes the hydrolysis of acetyl CoA to acetate and CoA side reaction

• Citrate synthase exhibits sequential, ordered kinetics

  • oxaloacetate induces a structural rearrangement that creates an acetyl CoA binding site

Citric Acid cycle Step 3

Citrate is isomerized into isocitrate

• iron-sulfur protein (nonheme iron protein) = protein that contains iron that is not bonded to heme

  • ex. aconitase

• aconitase catalyzes the isomerization of citrate into isocitrate through a dehyrdation step and a hydration step

Citric Acid cycle step 4

Isocitrate is oxidized an decarboxylated to alpha-ketoglutarate

• isocitrate dehydrogenase catalyzes the oxidative decarboxylation of isocitrate, forming α-ketoglutarate and the high transfer-potential electron carrier NADH.

  • proceeds through the unstable oxalosuccinate

  • CO₂ is released from oxalosuccinate to yield α-ketoglutarate

    
    

Citric Acid cycle Step 5

Succinyl coenzyme A is formed by the oxidative decarboxylation of Alpha-ketoglutarate

• The α-ketoglutarate dehydrogenase complex catalyzes the oxidative decarboxylation of α-ketoglutarate to succinyl CoA, yielding NADH.

Citric Acid Cycle step 6

A compound with high phosphoryl-transfer potential is generated from succinyl coenzyme A

• succinyl CoA synthetase catalyzes the cleavage of a thioester linkage of succinyl CoA, yielding succinate

  • coupled to the phosphorylation of ADP or GDP because the ∆G°′ for the hydrolysis is comparable to that of ATP

  • The reaction is readily reversible

True or false: ATP or GTP formations may be coupled to the formation of succinate

True

• Mammals have two isozymic forms of the enzyme

• The GDP-requiring enzymes predominates in tissues performing anabolic reactions (ex. liver), and the GTP is used to power succinyl CoA synthesis

• the ADP-requiring enzyme predominates in tissues performing large amounts of cellular respiration )ex. skelatal and heart muscel

Citric Acid cycle step 7

Oxaloacetate is regenerated by the oxidation of succinate

• Succinate dehydrogenase, fumarase, and malate dehydrogenase catalyze successive reactions of four- carbon compounds to regenerate oxaloacetate.

• FADH2 and NADH are generated.

• Once regenerated, oxaloacetate can initiate another cycle.

Citric acid cycle step 8

Succinate is oxidized to fumarate by succinate dehydrogenase

• FAD is the hydrogen acceptor because the free-energy change is insufficient to reduce NAD⁺

Succinate dehydrogenase

• is an iron-sulfur protein has the isoalloxazine ring of FAD covalently attached to a histidine side chain

• is embedded in the inner mitochondrial membrane

• is directly associated with the electron-transport chain

Citric acid cycle step 9

Fumarate is hydrates to L-Malate by fumarase

• fumarase catalyzes the stereospecific trans addition of H⁺ and OH^-, yielding only the L-isomer of malate

Citric acid cycle step 10

Malate dehydrogenase catalyzed the oxidation of malate, yielding oxaloacetate and NADH

• ∆G°′ is significantly positive (∆G°′= +29.7 kJ mol−1).

• The reaction is driven by the use of the products: oxaloacetate by citrate synthase and NADH by the electron-transport chain.

Citric acid cycle net reaction

The two carbon atoms that enter each cycle as acetyl CoA are not the ones that leave as CO₂ during the initial two decarboxylation reaction

Citric acid cycle produces high transfer-potential electrons, ATP, and CO₂

Stoichiometry of the citric acid cycle

• Two carbon atoms enter in the form of acetyl CoA, and two carbons leave in the form of CO2 molecules.

• Four pairs of hydrogen atoms leave in four oxidation reactions (yielding three NADH and one FADH2).

• One compound with high phosphoryl-transfer potential (usually ATP) is generated.

• Two water molecules are consumed.

How many enzyme-catalyzed reactions make up the full citrix acid cycle?

8

• There is a physical association of the citric acid cycle enzymes into a supramolecular complex

  • allows for substrate channeling

Regulation of entry to the citric acid cycle and metabolism through it

• The formation of acetyl CoA from pyruvate is irreversible in animal cells.

• Acetyl CoA has two principal fates:

  • metabolism by the citric acid cycle

  • incorporation into lipids

• The activity of the pyruvate dehydrogenase complex is tightly controlled allosterically and by reversible phosphorylation.

Pyruvate dehydrogenase complex is regulated allosterically

• High concentrations of reaction products inhibit the reaction by informing the enzyme that there is no need to metabolize pyruvate to acetyl CoA

  • Acetyl CoA inhibits the transacetylase component (E₂)

  • NADH inhibits the dihydrolipoyl dehydrogenase (E₃)

• regulated by reversible phosphorylation

Regulation in biological conditions

• At rest, the ratios of NADH/NAD⁺, acetyl CoA/CoA, and ATP/ADP are high

  • promotes phosphorylation and inactivation of the complex by activating PDK

• during activity:

  • high ADP and pyruvate activate the complex by inhibiting the kinase

  • Ca²⁺ stimulates the phosphatase, enhancing the pyruvate dehydrogenase activity

Regulation in citric acid cycle

• Regulated at several points

• isocitrate dehydrogenase and α-ketoglutarate dehydrogenase are allosteric enzymes that primarily regulate the rate of cycling.

  • These are the first two enzymes that harvest high-energy electrons in the cycle.

    
    

How is the citric acid cycle replenished iv various components are consumed for other pathways?

• Citric acid cycle intermediates must be replenished if any are used for biosyntheses.

• Mammals lack the enzymes for the net conversion of acetyl CoA into oxaloacetate or other cycle intermediates.

• anaplerotic reaction = a reaction that leads to the net synthesis, or replenishment, of pathway components

• Pyruvate carboxylase catalyzes the formation carboxylation of pyruvate to oxaloacetate.

• This reaction is used in gluconeogenesis and is dependent on the presence of acetyl CoA.