Chapter 12 - The Citric Acid Cycle / Tricarboxylic Acid Cycle (TCA) / Krebs Cycle

Citric Acid Cycle (TCA Cycle)

• Amphibolic - catabolic & anabolic

• involved in aerobic catabolism of carbs, lipids, AA

• intermediates are starting points for many biosynthetic reactions

• enzymes of cycle are in mitochondria (in eukaryotes) or cytosol (in bacteria)

• energy of oxidation reactions is largely conserved as reducing power

  • coenzymes NAD and Uniquinone (Q) are reduced

Entry of Pyruvate into the Mitochondrion

• pyruvate translocase transports pyruvate into mitochondria in symport with H+

  • translocase is located in inner membrane of mitochondria

Pyruvate Dehydrogenase Complex (PDH Complex)

• multienzyme complex containing 3 enzymes + 5 coenzymes (+ ATP coenzyme as regulator)

• enzyme components

  • E1 = pyruvate dehydrogenase

  • E2 = dihydrolipoamide acetyltransferase

  • E3 = dihydrolipoamide dehydrogenase

• coenzyme components

  • Thiamine Pyrophosphate (TPP)

  • HS—CoA

  • FAD & NAD+

  • Lipoic Acid

  • ATP

Structure of PDH Complex

a) Core of complex has 24 E2 chains

b) model of entire complex:

• 12 E1 dimers (blue)

• 6 E3 dimers (green

Role of PDH Complex Enzymes

• NAD⁺ & HS—CoA are cosubstrates (loosely bound)

• TPP, lipoamide, FAD are prosthetic groups (tightly bound)

• ATP is a regulator

• Lipoamide on E2 transfers the 2 C unit from E1 active site to E3 active site (substrate channeling)

Steps of PDH Complex

Step 1: Pyruvate + TPP + H⁺ —pyruvate dehydrogenase→ Hydroxyethylthiamine pyrophosphate (HETPP) + CO2

Step 2 (also catalyzed by E1): HETPP + Lipoamide → Acetyl-TPP + Dihydrolipoamide + H⁺ → Ylid + Acetyl-dihydrolipoamide

Step 3: Acetyl-dihydrolipoamide + HS—COA → Dihydrolipoamide + Acetyl CoA

Step 4: Dihydrolipoamide + E3—FAD → Lipoamide + E3—FADH₂

Step 5: E3—FADH2 + NAD⁺ → E3—FAD + NADH + H⁺

Step 1 of TCA Cycle

Acetyl CoA + Oxaloacetate + H2o → Citrate + HS—CoA + H⁺

• entry of substrate by condensation with oxaloacetate using citrate synthase

• irreversible

• carboxyl group of aspartate attacks methyl group of acetyl-CoA which is then joined to C4 of oxaloacetate

Step 2 of TCA Cycle

Citrate ⇆ Isocitrate

• rearrangement using aconitase

  • type of isomerase

• elimination of H2O from citrate to form C=C bond of cis-aconitate

• stereospecific addition of H2O to cis-aconitate to form 2R,3S-Isocitrate

Step 3 of TCA Cycle

Isocitrate + NAD⁺ → α-Ketoglutarate + NADH + CO₂

• first oxidative decarboxylation of isocitrate to α-ketoglutarate (α-kg) using isocitrate dehydrogenase

• irreversible

• ¼ oxi-red reactions

• hydride ion from C2 of isocitrate is transferred to NAD⁺ to make NADH

Step 4 of TCA Cycle

α-Ketoglutarate + HS—CoA + NAD⁺ → Succinyl CoA + NADH + CO₂

• second oxidative decarboxylation using α-ketoglutarate dehydrogenase complex

  

• irreversible

Structure of α-kg Dehydrogenase Complex

• similar to pyruvate dehydrogenase complex

• same coenzymes, identical mechanism

  • E1 - α-kg dehydrogenase (with TPP)

  • E2 - succinyltransferase (with flexible lipoamide prosthetic group)

  • E3 - dihydrolipoamide dehydrogenase (with FAD)

Step 5 of TCA Cycle

Succinyl CoA + GDP (or ADP) + P_i ⇆ Succinate + GTP (or ATP) + HS—CoA

• substrate-level phosphorylation using succinyl-CoA synthetase

• free energy in thioester bond of succinyl CoA is conserved as GTP (or ATP in plants, some bacteria)

Step 6 of TCA Cycle

Succinate + Q ⇆ Fumarate + QH₂

• oxidation using succinate dehydrogenase complex (SDH)

  • located on inner mitochondrial membrane

  • dehydrogenation is stereospecific; only trans isomer formed

  • substrate analog malonate is competitive inhibitor of SDH complex

Malonate

• structure analog of succinate

Structure of SDH Complex

• complex of several polypeptides, FAD prosthetic group, iron-sulfur clusters

• electrons are transferred from succinate to ubiquinone (Q), a lipid-soluble mobile carrier of reducing pwoer

• FADH2 generated is reoxidized by Q

• QH2 is released as mobile product

Step 7 of TCA Cycle

Fumarate + H₂O ⇆ L-Malate

• hydration by fumarase

• stereospecific trans addition of water to double bond of fumarate to form L-malate

Step 8 of TCA Cycle

L-Malate + NAD⁺ ⇆ Oxaloacetate + NADH + H⁺

• oxidation by malate dehydrogenase

Summary of TCA Cycle

For each acetyl CoA:

• 2 molecules of CO2 are released

• coenzymes NAD+ and Q are reduced to NADH and QH2 respectively

• 1 GDP (or ADP) is phosphorylated to GTP (or ATP) respectively

  • GDP used in mammals, ADP used in yeast/bacteria

• initial acceptor molecule (oxaloacetate) is reformed

Energy Conservation in TCA Cycle

• energy is conserved in reduced coenzymes NADH, QH2, and GTP

• NADH & QH2 can be oxidized to produce ATP by oxidative phosphorylation

Reduced Coenzymes That Produce ATP

• 3 NADH

  • 1 NADH = 2.5 ATP

• 2 QH₂

  • 1 QH2 = 1.5 ATP

• 1 GTP (or ATP)

Complete oxidation of 1 Acetyl CoA = 10 ATP

Fate of NADH in Anaerobic Glycolysis

• NADH is produced by G3PDH reaction is reoxidized to NAD+ in pyruvate to lactate reaction

• NAD+ recycling allows G3PDH reaction (and glycolysis) to continue anaerobically

Fate of NADH in Aerobic Glycolysis

• glycolytic NADH is not reoxidized via pyruvate reduction but is available to fuel ATP formation

• glycolytic NADH (cytosol) must be transferred to mitochondria (electron transport chain location)

• 2 NADH shuttles are available

NADH Shuttles

Malate-Aspartate Shuttle (most common)

• 1 Cytosolic NADH yields ~2.5 ATP

• total 32 ATP/glucose

Glycerol Phosphate Shuttle

• 1 Cytosolic NADH yields ~1.5 ATP

• total 30 ATP/glucose

Regulation of TCA Cycle

• allosteric modulators

• covalent modification of cycle enzymes

• supply of acetyl CoA

• regulation of pyruvate dehydrogenase complex controls acetyl CoA supply

Regulation of E2 & E3 in PDH Complex

• increased levels of Acetyl CoA & NADH inhibit E2 & E3 in mammals and E. coli

Regulation of Mammalian E1 in PDH Complex by Covalent Modification

Phosphorylation / Dephosphorylation of E1

Regulation of PDH Complex by PDK & PDP

Pyruvate Dehydrogenase Kinase (PDK)

• activated by NADH and Acetyl CoA, leading to inactivation of PDH complex

• inhibited by pyruvate and ADP, leading to activation of PDH complex

Pyruvate Dehydrogenase Phosphatase (PDP)

• stimulated by Ca²⁺, leading to activation of PDH complex

Regulation of Isocitrate Dehydrogenase (ICDH)

Mammalian ICDH

• allosteric effectors: activation with Ca²⁺ & ADP, inhibition with NADH

E. Coli ICDH

• bifunctional enzyme is reciprocally regulated by intermediates of glycolytic and TCA cycles

Anaplerotic Reactions

replenishment of intermediates in a metabolic pathway

• ex: pyruvate carboxylase in mammals, phosphoenolpyruvate carboxylase in plants and bacteria

Glyoxylate Cycle

• pathway for formation of glucose from noncarbohydrate precursors (acetyl CoA or any of its precursors) in plants, bacteria, ad yeast (not animals)

• leads from 2-C compounds to glucose

  • ethanol or acetate can be metabolized to acetyl CoA, then glucose

  • stored in seed oils in plants are converted to carbs during germination

  • can go from isocitrate to succinate or glyoxylate (skips steps 3-5) using isocitrate lyase

• in animals, acetyl CoA is not a C source for net formation of glucose