Enzymatic Steps of the Citric Acid Cycle: Steps 5 through 8

Overview of the Final Stages of the Citric Acid Cycle

• The Citric Acid Cycle is explored through its final four enzymatic steps, specifically steps five through eight.

• These steps are catalyzed by the following enzymes:

  • Succinyl CoA synthetase

  • Succinate dehydrogenase

  • Fumarase

  • Malate dehydrogenase

• A critical distinction is made between the first half and the second half of the cycle:

  • The first four steps contain three enzymatic reactions that are considered irreversible.

  • The final four steps (steps five through eight) consist entirely of reversible steps.

• The cycle left off at the alpha ketoglutarate dehydrogenase complex, which facilitates the oxidative decarboxylation of alpha ketoglutarate to succinyl CoA.

• Succinyl CoA contains a high-energy thioester bond introduced via the addition of coenzyme A.

• This high-energy thioester bond provides the necessary energy to be captured as electron carriers and $GTP$ in the subsequent four steps.

• These final four steps are dedicated to extracting the energy stored within the high-energy succinyl CoA molecule.

Step 5: Succinyl CoA Synthetase

• Reaction Overview: This step involves the conversion of succinyl CoA into succinate.

• Reaction Formula: $\text{Succinyl CoA} + P_i + \text{GDP} \rightarrow \text{Succinate} + \text{CoA} + \text{GTP}$

• Substrate Level Phosphorylation: This reaction is classified as a substrate level phosphorylation because a single enzyme step leads to the formation of a high-energy phosphoanhydride bond.

• Mechanism Details:

  • The substrate is succinyl CoA.

  • The product is succinate.

  • Coenzyme A (specifically reduced coenzyme A) is released from the substrate.

  • The breaking of the high-energy thioester bond in succinyl CoA drives the phosphorylation of $GDP$ (guanosine diphosphate) to form $GTP$ (guanosine triphosphate), which is structurally similar to $ATP$.

• Comparison to Glycolysis: Substrate level phosphorylation also occurs in two steps of glycolysis:

  • Step 7: Catalyzed by phosphoglycerate kinase (phosphorylation of $ADP$ to $ATP$).

  • Step 10: Catalyzed by pyruvate kinase (phosphorylation of $ADP$ to $ATP$).

  • The succinyl CoA synthetase step is viewed as the third instance of substrate level phosphorylation within the broader process of cellular respiration.

• Organismal Variations:

  • In mammalian cells: The cycle primarily phosphorylates $GDP$ to produce $GTP$.

  • In plants: The reaction typically involves the phosphorylation of $ADP$ to produce $ATP$.

• Thermodynamics and Coupling:

  • The Gibbs free energy change ($\Delta G$) for the succinyl CoA synthetase reaction is close to zero, making it reversible.

  • The energy driving this reaction comes from two sources: the breaking of the thioester bond and the highly negative $\Delta G$ of the preceding alpha ketoglutarate dehydrogenase reaction.

  • These two enzymatic steps are considered to be coupled to drive the substrate level phosphorylation event.

• Nucleoside Diphosphate Kinase:

  • There is an enzyme called nucleoside diphosphate kinase that can convert $GTP$ to $ATP$.

  • The reaction is: $\text{GTP} + \text{ADP} \rightleftharpoons \text{GDP} + \text{ATP}$

  • The $\Delta G$ for this conversion is $0$, representing a neutral energy change.

  • Because of this enzyme, the yield of $GTP$ in the citric acid cycle is considered equivalent to the formation of $ATP$.

Step 6: Succinate Dehydrogenase

• Reaction Overview: This step involves the conversion of succinate to fumarate via a dehydrogenation reaction.

• Reaction Formula: $\text{Succinate} + \text{FAD} \rightarrow \text{Fumarate} + \text{FADH}_2$

• Electron Transfer: Succinate loses electrons and two protons to the electron carrier $FAD$ (flavin adenine dinucleotide), producing $FADH_2$.

• Thermodynamics: The Gibbs free energy change for this reaction is $0\,kJ\,mol^{-1}$, indicating it is a neutral energy change and a reversible reaction.

• Unique Localization:

  • While most citric acid cycle enzymes are soluble and located in the mitochondrial matrix, succinate dehydrogenase is embedded in the inner mitochondrial membrane.

  • It is the only enzyme of the cycle not located freely within the matrix.

• Dual Functionality:

  • Succinate dehydrogenase serves a dual role: it is a component of the citric acid cycle and also acts as part of the mitochondrial electron transfer chain (Complex II) in oxidative phosphorylation.

• Prosthetic Group and Vitamins:

  • The enzyme uses $FAD$ as an electron acceptor instead of $NAD^+$.

  • $FAD$ is a prosthetic group, meaning it is tightly bound or permanently associated with the enzyme.

  • This enzyme requires Riboflavin (Vitamin $B_2$) as a precursor to synthesize the $FAD$ coenzyme.

  • Proteins containing an $FAD$ prosthetic group are categorized as flavoproteins.

• Inhibition by Malonate:

  • Malonate is a structural analog of succinate, differing only by the absence of one carbon group.

  • Malonate acts as a competitive inhibitor; it binds to the active site of succinate dehydrogenase, preventing succinate from binding and inhibiting enzyme activity.

  • Malonate occurs naturally in some fruits and vegetables at very low levels, posing no significant health risk.

  • Historically, malonate was critical for research to understand the structure and reaction mechanism of the succinate dehydrogenase complex.

Step 7: Fumarase

• Reaction Overview: Fumarate undergoes a hydration reaction to produce malate.

• Reaction Formula: $\text{Fumarate} + \text{H}_2\text{O} \rightarrow \text{Malate}$

• Mechanism:

  • The reaction involves the input of a water molecule ($H_2O$).

  • It passes through a transition state characterized by the addition of a hydroxyl group followed by the addition of a proton.

• Thermodynamics: The Gibbs free energy change is close to zero, making it a readily reversible reaction in isolation.

Step 8: Malate Dehydrogenase

• Reaction Overview: This is the final step of the cycle, involving the dehydrogenation of malate to regenerate oxaloacetate.

• Reaction Formula: $\text{Malate} + \text{NAD}^+ \rightarrow \text{Oxaloacetate} + \text{NADH} + \text{H}^+$

• Electron Transfer: Hydrogen ions and electrons are removed from malate and donated to $NAD^+$ to generate $NADH$ and a free proton.

• Thermodynamic Challenge:

  • This reaction has a very highly positive Gibbs free energy change ($\Delta G$).

  • Under standard conditions, the reaction is very unfavorable in the forward direction.

  • The equilibrium constant ($K_{eq}$) would normally favor the accumulation of the substrate, malate, over the product, oxaloacetate.

• Driving the Reaction Forward:

  • In the mitochondria, this reaction proceeds forward because oxaloacetate is rapidly consumed by the first step of the cycle (citrate synthase).

  • Citrate synthase performs an irreversible condensation of acetyl-CoA and oxaloacetate, effectively "pulling" the malate dehydrogenase reaction forward by keeping oxaloacetate concentrations extremely low.

  • This irreversible nature of the first step provides the driving force for all the reversible reactions from succinyl CoA through to citrate.

Modern Research Techniques: NMR Spectroscopy

• While original research into Citric Acid Cycle substrates used muscle tissue homogenates, modern technology utilizes NMR (Nuclear Magnetic Resonance) spectroscopy.

• This allows researchers to observe the flux through the citric acid cycle within intact cells, tissues, and organisms.

• Methodology:

  • Precursors are labeled with heavy carbon isotopes, specifically $C^{13}$ instead of the common $C^{12}$.

  • These radiolabeled precursors are fed into the tissue.

  • The movement of the $C^{13}$ label through the various intermediates of the cycle is tracked.

• Applications: In modern medicine, NMR spectroscopy is used to evaluate the functionality of the citric acid cycle in clinical settings.