Citric Acid Cycle steps

Chapter 19: The Tricarboxylic Acid Cycle

• Chapter 19 covers the Tricarboxylic Acid Cycle (TCA cycle).
• Important questions addressed are how pyruvate is oxidized under aerobic conditions and the chemical logic governing this process.
• The summary of the chapter covers the chemical logic, the oxidative decarboxylation of pyruvate to acetyl-CoA, CO2 production from acetyl-CoA, regeneration of oxaloacetate, the energetic consequences, intermediates for biosynthesis, anaplerotic reactions, and regulation of the cycle.

History of the TCA Cycle

• In 1932, Krebs discovered that small acids like succinic, fumaric, malic, citric, oxaloacetic, and acetic become oxidized in kidney and liver tissues.
• In 1935, Albert Szent-György found that 4-carbon acids in pigeon muscle leads to the consumption of more O2 is consumed than required by those acids alone.
• Scientists concluded that the addition of acid activated the burning of substances like glucose and adding enzyme inhibitors revealed crucial steps in oxidation.
• Martius and Knoop showed that citrate converts to isocitrate.
• Krebs showed that oxaloacetate becomes citrate in muscle tissue, postulating his 2nd cycle (urea cycle in 1932).

Glucose Oxidation

• The oxidation of glucose to CO2 is a 24-electron oxidation.
• Electrons derived from glucose oxidation feed into the electron transport pathway, which drives ATP synthesis.
• Pyruvate from glycolysis is oxidatively decarboxylated to acetate.
• Acetate then degrades to 2 CO2 in the cycle with some ATP produced and more NADH.
• NADH contributes to ATP production via electron transport and oxidative phosphorylation.
• The TCA cycle is also known as the Krebs cycle or the citric acid cycle.

Chemical Logic of the TCA Cycle

• The TCA cycle oxidizes acetate units to CO2. Oxidation of acetyl requires C-C cleavage.
• C-C bond cleavage generally includes cleavage between carbons α- and β- to a carbonyl or α-cleavage of an α-hydroxyketone. These cleavages aren't effective for acetyl-CoA.
• Condensing acetate with oxaloacetate allows for β-cleavage.
• This cleavage combines with oxidation to form CO2, regenerating oxaloacetate, capturing energy as NADH and ATP.

Entry into the TCA Cycle

• Pyruvate converts to acetyl-CoA only in mitochondria, but in the cytosol, AcCoA sources are from citrate or amino acid degradation, not pyruvate.
• Pyruvate dehydrogenase utilizes TPP, COASH, lipoic acid, FAD, and NAD+.
• Citrate synthase (CS) is classic CoA chemistry.
• NADH and succinyl-CoA are allosteric inhibitors.

Pyruvate Dehydrogenase

• Pyruvate Dehydrogenase is a 3 enzyme complex (PDC): 2 dehydrogenases and one transacylase.
• The complex includes PDH (pyruvate dehydrogenase), dihydrolipoyl transacetylase (TA), and dihydrolipoyl dehydrogenase (DLD).
• Pyruvate loses CO2 and gets converted into HETPP.
• A hydroxyethyl group transfers to lipoic acid and oxidizes to form acetyl dihydrolipoate.
• The acetyl group finally transfers to CoA, reoxidizing lipoic acid.
• Decarboxylation of pyruvate yields hydroxyethyl-TPP.

Reactions of the TCA Cycle

• Each reaction has a specific enzyme

Citrate Synthase

• Citrate synthase initiates the TCA cycle, forming citrate from oxaloacetate and acetyl-CoA via a nucleophilic attack.
• Hydrolysis of this thioester drives the reaction.
• Citrate synthase has a Keq1=1, but its large AG°=-31.4 KJ/mol, drives it and is basically irreversible.
• AcCoA anion is protected from H2O in an hydrophobic pocket.
• NADH is an allosteric inhibitor due to the large AG0.
• The enzyme in mammals is a dimer of 49-kD subunits, with active sites lying in a cleft between two domains, surrounded by α-helical segments

Aconitase

• Aconitase isomerizes citrate to isocitrate, changing a tertiary alcohol to secondary -OH.
• Aconitase removes the pro-R H of the pro-R arm of citrate.
• It holds an iron-sulfur cluster prosthetic group.
• This reaction converts citrate to cis-aconitate, then to isocitrate.

Flouroacetate

• Fluoroacetate is a highly poisonous agent that blocks the TCA cycle in vivo by inhibiting aconitase.
• Fluoroacetate is traced to aconitase, inhibits fluorocitrate.
• Aconitase utilizes an iron-sulfur cluster. Binding of Fe2+ to the cluster vacant position activates aconitase.
• The added iron atom coordinates the C-3 carboxyl and hydroxyl groups of citrate and acts as a Lewis acid, accepting an electron pair from the hydroxyl group and making it a better leaving group.

Isocitrate Dehydrogenase

• A key step is oxidative decarboxylation transforming isocitrate into α-ketoglutarate via classic NAD+ chemistry.
• This enzyme links the cycle to the electron transport pathway
• Reaction occurs by oxidizing the C-2 alcohol of isocitrate to form oxalosuccinate, with decarboxylation expelling CO2.
• ADP makes Km for isocitrate 10x smaller, while ATP and NADH are allosteric inhibitors.

α-Ketoglutarate Dehydrogenase

• α-Ketoglutarate dehydrogenase catalyzes the second oxidative decarboxylation, nearly identical in structure and mechanism to pyruvate dehydrogenase and uses five coenzymes
• The reaction is a multienzyme complex.

Substrate-Level Phosphorylation

• Succinyl-CoA synthetase catalyzes reactions through phosphorylation
• It involves the hydrolysis of succinyl-CoA, which drives GDP phosphorylation, utilizing a phosphohistidine intermediate

Conversion of Succinate to Oxaloacetate

• It involves is crucial as it will be seen in other reactions such as fatty acid biosynthesis.
• The reactions entail an oxidation of a C-C single bond to a double bond, hydration across the double bond, and oxidation of the resulting alcohol to a ketone.

Succinate Dehydrogenase

• This reaction requires Succinate Dehydrogenase Is FAD-Dependent (FAD oxidation of a single bond to a double bond).
• It involves hydride removal by FAD/deprotonation and is part of the electron transport pathway in the mitochondrial membrane.
• Electrons are transferred from succinate to FAD, passing directly to ubiquinone (UQ) in the electron transport pathway.
• FAD is covalently bound.

Fumarase and Malate Dehydrogenase

• Fumarase catalyzes the trans-hydration of fumarate to form L-malate.
• Hydration occurs across the newly formed double bond via trans-addition.
• Malate dehydrogenase completes the cycle by oxidizing malate to oxaloacetate.
• It's energetically costly (AG°' = +30 kJ/mol), resulting in low oxaloacetate concentrations.

Energetic Consequences

• One acetate molecule produces two CO2, one ATP, and four reduced coenzymes.
• The TCA cycle is exergonic, with ∆G°' of -40 kJ/mol.
• Combining glycolysis and TCA yields twelve reduced coenzymes, potentially producing 34 ATP molecules

Fate of Carbon Atoms

• The carbon atoms of acetyl-CoA have different fates in the TCA Cycle.
• The carbonyl C of acetyl-CoA becomes CO2 in the second cycle turn.
• The methyl C of acetyl-CoA survives two cycles, with half exiting each turn thereafter.

Biosynthesis

• The TCA cycle provides several intermediates:
  • α-Ketoglutarate: Makes glutamate for building nucleotides, arginine, and proline.
  • Succinyl-CoA: Used in porphyrin synthesis.
  • Fumarate and oxaloacetate: Makes nucleotides and amino acids.
  • Citrate: Exports from mitochondria to act as acetyl-CoA and oxaloacetate in the cytoplasm.

Anaplerotic Reactions

• Anaplerotic Rxns “fill up” the cycle.
• Pyruvate carboxylase converts pyruvate to oxaloacetate.
• PEP carboxylase converts PEP to oxaloacetate, and malic enzyme converts pyruvate to malate.

Reductive TCA Cycle

• The reductive TCA cycle runs backward, assimilating to serve bacterial carbon needs and may have been the first metabolic pathway.
• A potential energy source is the reaction of FeS with H₂S to form FeS2 (iron pyrite), where iron pyrite is a version of iron-sulfur clusters.

Regulation

• Three key reactions, citrate synthase (inhibited by ATP, NADH and succinyl-CoA), isocitrate dehydrogenase (inhibited by ATP, activated by ADP and NAD+), and α-Ketoglutarate dehydrogenase (inhibited by NADH and succinyl-CoA, activated by AMP), are the key regulation sites.
• Pyruvate dehydrogenase regulation includes activation by NAD+ and CoA and inhibition by ATP, NADH, and acetyl-CoA .
• Pyruvate Dehydrogenase is also regulated by phosphorylation.

TCA Cycle as a Metabolon

• Self-assembly concentrates enzymes, with enzymes passing substrates to each-other which increases flux and efficiency.
• Positively charged surfaces helps transport the negative substrates.

Glyoxylate Cycle

• The glyoxylate cycle occurs because TCA cycle-based growth cannot do net synthesis of carbohydrates from acetate.
• The glyoxylate cycle bypasses CO2-evolving steps, utilizing extra acetate through isocitrate lyase and malate synthase, which are short circuiting enzymes.
• Seeds rely on this pathway until photosynthesis begins.
• Isocitrate lyase yields glyoxylate and succinate.
• Malate synthase facilitates a Claisen condensation between acetyl-CoA and glyoxylate using CoA chemistry.

Questions about the Citric Acid Cycle. Topics include: hydrophobic pocket in citrate synthase, aconitase isomerization, fluoroacetate inhibition, succinate dehydrogenase reaction, and the role of FAD and NADH.