Krebs Cycle: Citric Acid Cycle

• The topic is the Krebs cycle, also known as the citric acid cycle.

Krebs Cycle Overview

• Oxygen is used by cells as an electron acceptor in the Krebs cycle.
• The oxidative metabolism of biomolecules occurs in three stages.
• The first stage involves breaking down macromolecules into smaller pyruvate molecules, which are then broken down into Acetyl-CoA molecules; this stage includes glycolysis.
• The second stage is the Krebs cycle, which involves fully oxidizing the carbon atoms from Acetyl-CoA into CO2 molecules, releasing energy in the form of nucleotide triphosphates (GTP) and reducing molecules (FADH2 and NADH2).
• The third stage involves the electron transport chain and oxidative phosphorylation, where the reducing power generated in the Krebs cycle synthesizes ATP.
• Polysaccharides, monosaccharides, proteins, amino acids, and lipids can be oxidized completely in the Krebs cycle.
• It also involves amino acids being converted to pyruvate or Acetyl-CoA.
• It takes place within the mitochondria matrix.
• For prokaryotes, the reactions of the Krebs cycle occur in the cytosol.

Krebs Cycle Steps

• The Krebs cycle involves eight fundamental steps that release CO2 and redox agents (NADH + H+ and FADH2).
• Eight enzymes are involved in the Krebs cycle: citrate synthase, aconitase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinyl-CoA synthetase, succinate dehydrogenase, fumarase, and malate dehydrogenase.
• The eight reaction steps are: condensation, dehydration and hydration, the first oxidative decarboxylation, the second oxidative decarboxylation, substrate-level phosphorylation, oxidation, hydration, and oxidation.

Formation of Citrate

• The Krebs cycle begins when Acetyl-CoA donates its two-carbon acetyl group to the 4-carbon oxaloacetate, resulting in condensation.
• The enzyme citrate synthase catalyzes this reaction, producing citrate (6C), which is an irreversible reaction.

Transformation of Citrate into Isocitrate

• Transformation of citrate into isocitrate occurs in two steps: dehydration that forms cis-aconitate and hydration that forms isocitrate.
• The enzyme aconitase catalyzes this reversible reaction.

First Oxidative Decarboxylation

• In this irreversible reaction, isocitrato (6C) becomes α-ketoglutarate (5C) by reducing NAD+ to NADH + H+ and removing a carbon atom as CO2.
• The enzyme isocitrate dehydrogenase catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate.

Second Decarboxylation

• The step is the oxidative decarboxylation of α-ketoglutarate (5C) into succinyl-CoA (4C) along with the release of a carbon atom in the form of CO2.
• The α-ketoglutarate dehydrogenase multi-enzyme complex carries out this irreversible reaction. α-ketoglutarate dehydrogenase is virtually identical to the pyruvate dehydrogenase (PDH) enzyme complex.
• NAD+ acts as an electron acceptor, producing NADH + H+.
• CoA-SH acts as a transporter of a succinyl group.

Phosphorylation

• A succinyl-CoA molecule with a high-energy thioester bond converts an ATP or GTP molecule in this step.
• A molecule of GTP is synthesized from GDP and Pi, releasing succinate and free coenzyme A (CoA-SH) where succinyl-CoA synthetase is the enzyme to facilitate the reaction.

Succinate Oxidation

• Succinate is transformed into fumarate by the flavoprotein succinate dehydrogenase.
• In a dehydrogenation reaction, the oxidation of the single bond at the center of the succinate molecule leads to a double trans bond, where the hydrogen eliminated couples to the synthesis of a molecule of FADH2 originating from FAD.
• All Krebs cycle enzymes are found in the mitochondrial matrix, except succinate dehydrogenase, which is found in the inner mitochondrial membrane.

Hydration of Fumarate

• Fumarase performs the reversible hydration of fumarate to L-malate.
• The enzyme is stereospecific and catalyzes the hydration of the double bond in trans of fumarate but not with the cis fumarate.

Oxidation of Malate

• L-malate dehydrogenase oxidizes L-malate to oxaloacetate, with NAD+ participation that results in the reduction of a third molecule of NAD+ to NADH + H+.
• The oxaloacetate is regenerated for use in the generation of citrate.

Energy Produced

• Oxidation reactions conserve energy in the form of NADH + H+ and FADH2, along with ATP or GTP formation.
• The ATP generated is only one ATP per cycle, however, redox reactions in the form of NADH + H+ and FADH2 enter the electron transport chain and produce a large number of ATP molecules during oxidative phosphorylation.

Balance

• One molecule of Acetyl-CoA is consumed and two molecules of CO2 are obtained in the complete cycle's turn.
• Acetyl-CoA + 3NAD+ +FAD GDP + Pi + 2H2O becomes 2CO2 + 3NADH + 3H+ +FADH2 + GTP + CoA-SH
• The breakdown of one molecule of Acetyl-CoA in the Krebs cycle generates three molecules of NADH + H+, one molecule of FADH2, one molecule of GTP, and two molecules of CO2.
• Starting with two molecules of Acetyl-CoA from two pyruvates generates six molecules of NADH + H+, two molecules of FADH2, two molecules of GTP, and four molecules of CO2.
• The last reactions prepare preparation for another cycle by regenerating oxaloacetate.
• The Krebs cycle can occur with a single molecule of oxaloacetate.

Regulation

• The Krebs cycle is strongly regulated to meet the cell's needs and is a source of precursors for biomolecules.
• Regulation primarily occurs at two levels: substrate availability and modulation of key enzymes.

Substrate Availability

• AcetylCoA and oxaloacetate exist in low concentrations in the mitochondria relative to citrate synthase. Availability of these stimulates citrate synthesis and the Krebs cycle.
• Acetyl-CoA availability affects pyruvate dehydrogenase (PDH)
• Inhibited by ATP, AcetylCoA, and NADH and activated by AMP, CoA-SH, and NAD.
• Oxalacetate's complex acts through pyruvate carboxylase.

Key Enzyme Modulation

• The key enzymes are citrate synthase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase.
• Citrate synthase is inhibited by succinyl CoA and NADH + H+.
• Isocitrate dehydrogenase is inhibited by ATP and NADH + H+.
• α-cetoglutarate dehydrogenase is inhibited by succinyl CoA and NADH + H+.

Additional Key Points

• Only glucose needed to supply the cycle is metabolized to pyruvate to meet energy needs.
• Glycolysis couples to the Krebs cycle through ATP and NADH but is also affected by citrate that inhibits PFK-1.

Anaplerotic Reactions and Amphibolic Character

• Krebs cycle molecules can be used by the cell for other purposes.
• Catabolic routes meet in the Krebs cycle and replenish the intermediates in the cycle.
• Anaplerotic reactions allow the cycle to continue functioning
• Citrate can be used in the biosynthesis of lipids.
• ATP-citrate lyase converts cytoplasmic citrate into Acetyl-CoA to obtain fatty acids.
• α-ketoglutarate is used in amino acid biosynthesis.
• Succinyl-CoA is a precursor in porphyrin synthesis to obtain hem groups and cytochromes.
• Oxalacetate is used in gluconeogenesis and to synthesize amino acids like aspartate.

Anaplerótic Reactions

• Pyruvate carboxylase enables the carboxylation of pyruvate from glycosis to oxalacetate and requires ATP.
• Malic enzyme involves CO2 carboxylation with the production of malate from pyruvate that enters the Krebs cycle, converting to oxalacetate.
• PEP carboxykinase involves the carboxylation of phosphoenolpyruvate in oxalacetate.
• Transaminases replenish oxalacetate and α-ketoglutarate.

Glyoxylate Cycle

• An alternative route is the metabolism of acetyl-CoA to obtain succinate that causes oxalacetate. It can produce glucose from oxalacetate.
• Allows the net fixation of carbon atoms from fatty acids into glucose.
• Animals cannot perform this conversion.
• Involves the cycle of Krebs enzymes (citrate synthase and aconitase) and enzymes exclusive to the glyoxylate cycle (isocitrate lyase and malate synthase).
• The oxaloacetate condenses with acetyl-CoA to citrate.
• The citrate is transformed to isocitrate.
• The isocitrate splits to succinate with glyoxylate. The enzyme is isocitrate lyase.
• The glyoxylate condenses with acetyl-CoA forming malate by the enzyme malate synthase.
• The succinate exits the forming oxalacetate that can be converted to glucose.
• Malate regenerates oxalacetate.