Detailed Enzymatic Mechanisms of the Citric Acid Cycle: Steps 1-4

Step 1: Citrate Synthase and the Entry of Acetyl CoA

• Citrate synthase catalyzes the first step of the citric acid cycle, acting as the entry point where acetyl CoA feeds into the metabolic pathway.

• Acetyl CoA is produced via the oxidative decarboxylation of pyruvate by the pyruvate dehydrogenase complex within the mitochondrial matrix, where it is readily available for citrate synthase.

• In this reaction, acetyl CoA (a $2$-carbon acyl group, essentially an acetate) is condensed with oxaloacetate (a $4$-carbon compound) derived from the previous turn of the cycle.

• The reaction yields citrate, a $6$-carbon compound, and releases reduced coenzyme A ($HS-CoA$) as an additional product.

• The release of coenzyme A involves the cleavage of a thioester bond, which is a very high-energy bond.

• Thermodynamic properties:

  • The reaction is highly energetically favorable with a very large and negative Gibbs free energy change ($Δ G$).

  • Under cellular conditions within the mitochondrial matrix, this step is essentially irreversible.

• Regulatory role:

  • Because it is irreversible, this step is a primary site of regulation.

  • Citrate levels within the mitochondrion serve as an indicator of flux through the cycle.

  • Citrate itself can act as a regulator, inhibiting or activating enzymes in other metabolic pathways, thereby coordinating various metabolic activities.

• Structural dynamics (Induced Fit):

  • Space-filling models and protein crystallography experiments demonstrate that the enzyme undergoes a significant conformational change upon substrate binding.

  • The binding of oxaloacetate must occur first at the active site; this binding induces a structural change in the protein that creates the binding site for acetyl CoA.

Step 2: Aconitase and the Production of Isocitrate

• The second enzymatic step is catalyzed by aconitase and is divided into two sequential sub-steps: Step $2A$ and Step $2B$.

• Sub-step $2A$ (Dehydration): Citrate is converted into an intermediate product called cis-aconitate through a dehydration reaction that removes water ($H_2O$).

• Intermediate release: cis-aconitate is considered an intermediary product because it remains bound to the enzyme and is not released into the matrix.

• Sub-step $2B$ (Rehydration): The same aconitase enzyme then rehydrates cis-aconitate to form the final product of this step, isocitrate.

• Thermodynamics and Pathway Flux:

  • This reaction has a fairly high and positive $Δ G$, meaning the reverse direction (isocitrate to citrate) is theoretically favored under normal conditions if the enzyme were isolated.

  • In the context of the citric acid cycle pathway, the reaction is pulled forward because isocitrate is rapidly and immediately utilized by the subsequent enzyme (isocitrate dehydrogenase).

  • This continuous removal of product maintains a low concentration of isocitrate, drawing the reaction toward the forward direction to re-equilibrate.

The Dual Function and Moonlighting Role of Aconitase

• Iron-Sulfur Center:

  • Aconitase contains an iron-sulfur ($Fe-S$) center that acts as a cofactor.

  • This cluster is tightly bound within the active site by sulfur atoms from cysteine residues and is directly involved in catalysis.

  • Because it is permanently and tightly bound, it is classified as a prosthetic group.

• Dependency on Iron:

  • The enzyme's catalytic activity is strictly dependent on the availability of iron within the cell. Conditions like anemia, which result in insufficient iron, can lead to the absence of the prosthetic group and loss of metabolic function.

• Cytosolic Isoform and "Moonlighting":

  • Beyond the mitochondrial version, there is a cytosolic isoform of aconitase.

  • Active Form (Iron present): It functions catalytically to generate isocitrate, which is used to produce $NADPH$ for anabolic reactions like fatty acid synthesis.

  • Regulatory Form (Iron absent): When iron levels are low, the enzyme loses its iron-sulfur cluster and undergoes a drastic conformational change.

  • In this altered state, the protein binds to messenger RNA ($mRNA$) for proteins involved in iron transport and storage.

  • Regulatory Effect: It inhibits iron storage and promotes iron mobilization/transport to increase the availability of iron for essential cellular functions.

  • Definition: Aconitase is one of the first discovered "moonlighting enzymes," meaning a single protein that performs two distinct functions depending on cellular conditions.

Step 3: Isocitrate Dehydrogenase

• Isocitrate dehydrogenase catalyzes the conversion of isocitrate into $α$-ketoglutarate.

• This step is an oxidative decarboxylation reaction and is irreversible.

• Reaction details:

  • A carboxylate group is released from isocitrate as carbon dioxide ($CO_2$).

  • This is one of the primary steps responsible for the release of carbon dioxide in the cycle.

  • The reaction involves an electron transfer to nicotinamide adenine dinucleotide ($NAD^+$), producing $NADH$ and a proton ($H^+$).

• Isoforms and Coenzymes:

  • Mitochondrial isoform: Specifically uses $NAD^+$ to generate $NADH$ (the focus of the citric acid cycle).

  • Cytosolic isoform: Utilizes $NADP^+$ to generate $NADPH$.

• Prosthetic Group: This enzyme requires manganese ($Mn^{2+}$) as a prosthetic group to facilitate the reaction.

Step 4: Alpha-Ketoglutarate Dehydrogenase Complex

• The fourth step involves the conversion of $α$-ketoglutarate ($5$ carbons) to succinyl CoA ($4$ carbons) via the $α$-ketoglutarate dehydrogenase complex ($KDHC$).

• Like the previous step, this is an oxidative decarboxylation reaction and is irreversible.

• Reaction mechanics:

  • A carboxylate group is released as $CO_2$.

  • Coenzyme A ($HS-CoA$) is a substrate that enters the reaction to form a new high-energy thioester bond in the product, succinyl CoA.

  • Electrons are transferred to reduce $NAD^+$ into $NADH$.

  • The energy released from the oxidation of the substrate is captured and stored in the thioester bond of succinyl CoA.

• Comparison with Pyruvate Dehydrogenase Complex ($PDC$):

  • The $α$-ketoglutarate dehydrogenase complex is virtually identical in mechanism and structure to the $PDC$ used to convert pyruvate to acetyl CoA.

  • Both complexes use five coenzymes: $FAD$, $NAD^+$, thiamine pyrophosphate ($TPP$), coenzyme A ($CoA$), and lipoate.

  • Both are multi-enzyme complexes consisting of three subunits: $E_1$, $E_2$, and $E_3$.

  • Shared Subunits: The $E_3$ subunit is exactly the same in both complexes (and in the branched-chain amino acid dehydrogenase complex used for isoleucine metabolism), originating from the same gene.

  • Unique Subunits: The $E_1$ and $E_2$ subunits are slightly different between the complexes to accommodate the different substrates ($3$-carbon pyruvate vs. $5$-carbon $α$-ketoglutarate) and products ($2$-carbon acetyl CoA vs. $4$-carbon succinyl CoA).

Summary of the First Four Steps

• The first four steps represent the first half of the citric acid cycle.

• Of these four steps, three are irreversible (citrate synthase, isocitrate dehydrogenase, and $α$-ketoglutarate dehydrogenase) and one is reversible (aconitase).

• All the irreversible, highly regulated steps of the cycle occur within this first half.