TCA Cycle Notes

Overview

The tricarboxylic acid cycle (TCA cycle), also known as the Krebs cycle or citric acid cycle, is a central series of chemical reactions for aerobic respiration in eukaryotes and many bacteria. It fully oxidizes glucose-derived pyruvate to carbon dioxide, extracting more energy than glycolysis alone. This process occurs within the mitochondria of eukaryotic cells, specifically in the mitochondrial matrix.

Anaerobic Glycolysis vs. Aerobic Respiration

Anaerobic glycolysis: Glucose+2ADP+2Pi→2Lactate+2ATP+2H2O+2H+Glucose+2ADP+2Pi\rightarrow2Lactate+2ATP+2H2O+2H^{+}

Aerobic respiration yields significantly more ATP compared to anaerobic glycolysis through the complete oxidation of glucose.

Goal of the TCA Cycle

The primary goal is to oxidize acetyl-CoA (derived from carbohydrates, fats, and proteins) to produce reduced electron carriers (NADH and FADH2).

These carriers donate electrons to the electron transport chain (ETC) for oxidative phosphorylation, which is the main ATP-producing process in aerobic respiration.


The general reaction is:
Substrate⋅H2(reduced)+Acceptor(NAD+orFAD)→Substrate(oxidized)+Acceptor⋅H2(NADHorFADH2)Substrate \cdot H2 (reduced) + Acceptor (NAD^+ or FAD) \rightarrow Substrate (oxidized) + Acceptor \cdot H2 (NADH or FADH_2)

Mitochondria

  • Origin: Evolved from prokaryotic cells via endocytosis.

  • Genome: Contains its own genome encoding 13 genes for ETC subunits.

  • Membranes: Double membrane-bound (inner and outer).

  • Permeability: The inner membrane is impermeable to charged molecules (e.g., protons).

  • Cristae: The inner membrane is folded into cristae, increasing surface area for ETC and ATP synthase complexes.

  • Location of TCA Cycle: Inner mitochondrial matrix.

Pyruvate Transport

Pyruvate is actively transported into the mitochondrial matrix for oxidation.

  • Mitochondrial Pyruvate Carrier (MPC): Mediates pyruvate transport.

  • Symport with Protons: Pyruvate transport is coupled with proton transport.

  • pH Gradient: The matrix pH is approximately 7.8; the intermembrane space pH is 7.0-7.4.

  • Proton transport powers pyruvate transport down the concentration gradient.

MPC Downregulation and Glycolytic Flow

  • Differentiated Tissue: Primarily oxidative phosphorylation, producing approximately 36 ATP/glucose.

  • Proliferative Tissue: Increased glycolytic flow even in the presence of oxygen (Warburg effect).

  • Warburg Effect (Aerobic Glycolysis): Increased glucose uptake and lactate production in cancer cells, producing approximately 4 ATP/glucose.

  • Anaerobic Glycolysis: Produces 2 mol ATP/mol glucose.

Sources of Acetyl-CoA

Acetyl-CoA links multiple metabolic pathways.

  • Beta-oxidation: Fatty acid catabolism.

  • Amino Acid Catabolism: Isoleucine, leucine, and threonine.

  • Other amino acids enter the pathway at different points.

Pyruvate Dehydrogenase Complex (PDC)

PDC converts pyruvate into acetyl-CoA, linking glycolysis to the TCA cycle.

  • Subunits: E1 (Pyruvate dehydrogenase), E2 (Dihydrolipoyl transacetylase), E3 (Dihydrolipoyl dehydrogenase).

  • Size and Structure: Approximately 400 Å sphere with a molecular weight of ≈7800 kDa.

  • Ratios: Uneven ratios of E1, E2, E3 (22:60:6).

  • E2 Structure: 20 trimers with channels for acetyl group movement.

PDC Overview 1

  • Reactants: Pyruvate, CoA-SH

  • Products: Acetyl-CoA, CO2

  • Coenzymes: TPP, Acyl lipoyllysine, FAD, NAD+

PDC Overview 2

  • E1: Converts pyruvate to hydroxyethyl-TPP, releasing CO2.

  • E2: Transfers the acetyl group from hydroxyethyl-TPP to lipoamide, forming acetyl-dihydrolipoamide.

  • E3: Reoxidizes dihydrolipoamide.

Pyruvate Decarboxylase and TPP

Pyruvate decarboxylase uses thiamine pyrophosphate (TPP).

  • TPP Function: Produces a stable carbanion.

  • Role: Cleavage of bonds adjacent to a carbonyl group.

Coenzyme and Prosthetic Group Roundup

  • Thiamine pyrophosphate (TPP): Bound to E1; decarboxylates pyruvate.

  • Lipoic acid: Linked to Lys on E2; accepts the hydroxyethyl carbanion.

  • Coenzyme A (CoA): Substrate for E2; accepts the acetyl group.

  • Flavin adenine dinucleotide (FAD): Bound to E3; reduced by lipoamide.

  • Nicotinamide adenine dinucleotide (NAD+): Substrate for E3; reduced by FADH2.

Pyruvate Dehydrogenase Mechanism

  • E1: Decarboxylation (Reaction 1) and Hydroxyethyl group transfer (Reaction 2).

E2's Swinging Arm

The lipoyllysyl arm of E2 transfers intermediates between active sites.

Pyruvate Dehydrogenase Mechanism (cont.)

  • E2: Transesterification (Reaction 3).

  • E3: Reoxidation of dihydrolipoamide (Reaction 4) and Reoxidation of E3 (Reaction 5).

Regulation of PDC

PDC commits pyruvate to energy production. Activation/inactivation by kinase and phosphatase, regulated by allosteric factors.

Step 1 TCA: Citrate Synthase (and Regulation)

  • Reactants: Oxaloacetate + Acetyl-CoA

  • Product: Citrate

  • First irreversible, committed step.

  • Regulation: Inhibited by ATP, NADH, citrate, and succinyl-CoA.

Citrate Synthase Mechanism

  • Step 1: Forming enolate (nucleophile).

  • Steps 2 & 3: Enolate attack, break thioester.

Step 2 TCA: Aconitase

  • Stereo-specific isomerization.

  • Uses an Iron-Sulphur cofactor.

  • Secondary role in iron homeostasis when iron is depleted.

Fluoroacetate Poisoning

  • Fluoroacetate is a deadly poison.

  • Inhibits aconitase, blocking the TCA cycle.

Step 3 TCA: Isocitrate Dehydrogenase

  • Reactant: Isocitrate

  • Product: α-Ketoglutarate

  • Gain of an NADH.

  • Rate-limiting step.

  • Regulation: Inhibited by ATP and NADH.

Isocitrate Dehydrogenase Mechanism

  • Hydride transfer to NAD+.

  • Rearrangement of enol intermediate.

  • Mn2+ removes electrons, allowing for decarboxylation.

Step 4 TCA: α-Ketoglutarate Dehydrogenase

  • Gain of an NADH.

  • Irreversible step.

  • Large complex, similar to PDC.

  • Same mechanism of reaction with E1, E2, and E3.

  • Requires same coenzymes.

  • Regulation: Inhibited by NADH and Succinyl-CoA.

Step 5 TCA: Succinyl-CoA Synthetase

  • Reactant: Succinyl-CoA

  • Product: Succinate

  • Substrate-level phosphorylation of GDP (GTP is ATP energy equivalent).

  • Powered by breaking the thioester bond.

Succinyl-CoA Synthetase Mechanism

  • Step 1: Succinyl-CoA + Pi -> Succinyl-phosphate + COASH

  • Step 2: Succinyl-phosphate + Enzyme-His -> Succinate + 3-Phospho-His

  • Step 3: 3-Phospho-His + GDP -> Enzyme-His + GTP

Step 6 TCA: Succinate Dehydrogenase

  • Reactant: Succinate

  • Product: Fumarate

  • FADH2 reduced through reaction.

  • Enzyme is bound to the inner membrane.

  • Part of the electron transport chain as well as TCA (Complex II)!

Succinate Dehydrogenase & ETC

  • Complex II

Electron transfer in succinate dehydrogenase:

  • Electrons carried by FADH2 and three iron-sulphur clusters.

  • Iron-Sulphur clusters coordinated with cysteine residues.

  • This complex is integral to the TCA cycle as it facilitates the conversion of succinate to fumarate, contributing to the overall production of reducing equivalents for ATP synthesis.

Electron transfer in succinate dehydrogenase

  • Electrons carried by FADH2 and three iron-sulphur clusters.

  • Iron-Sulphur clusters coordinated with cysteine residues.

Step 7 & 8 TCA: Fumarase & Malate Dehydrogenase

  • Fumarate → Malate (Fumarase)

  • Malate → Oxaloacetate (Malate dehydrogenase)

Anaplerosis & Cataplerosis

  • Anaplerosis: Replenishes TCA cycle intermediates.

  • Cataplerosis: Draws down TCA cycle intermediates.

Anaplerosis & Cataplerosis (pathways)

  • Anaplerotic: Pyruvate to Oxaloacetate, Glutamate/Glutamine to α-Ketoglutarate, Histidine/Proline/Arginine to α-Ketoglutarate, Succinyl CoA to Succinate.

  • Cataplerotic: Oxaloacetate to Aspartate/Asparagine, Citrate to Fatty acids, α-Ketoglutarate to Glutamate/Glutamine, Fumarate to Phenylalanine/Tyrosine.

Pyruvate Carboxylase

  • Catalyzes the first step of gluconeogenesis.

  • Replenishes oxaloacetate.

  • Allosterically activated by acetyl-CoA.

  • Uses biotin as a prosthetic group.

Carbon Tracing

  • Radio labeling carbons (14C) to probe pathways.

  • Example: radio labeling of glucose.

  • Glycolysis splits glucose into two pyruvates.

  • Labeled carbons in pyruvate create consequences in TCA.

Carbon Tracing - Round 1 → 2 transition

Note carbons lost as CO2 and transitions between cycles. Acetyl-CoA and oxaloacetate combine to form citrate.

  • 14C-2 or 14C-5 glucose: Round 1: 100% radioactivity stays in the cycle, split between 2 carbons (50% each).

  • 14C-2 or 14C-5 glucose: Round 2: 100% radioactivity leaves the cycle as CO2.

  • 14C-3 or 14C-4 glucose: Round 1: 100% radioactivity leaves the cycle as CO2.

  • 14C-1 or 14C-6 glucose: Round 1: 100% radioactivity stays in cycle, split between 2 carbons (50% each).

  • 14C-1 or 14C-6 glucose: Round 2: 100% radioactivity stays in cycle, split between 4 carbons (25% each).

  • 14C-1 or 14C-6 glucose: Round 3: 50% radioactivity stays, 50% leaves.

  • 14C-1 or 14C-6 glucose: Round 4: 50% radioactivity stays, 50% leaves.
    Round 3’s pattern repeats FOREVER.