The Kreb Cycle: A Detailed Explanation of its Reactions and Regulation, Lecture notes of Metabolic Nutrition

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The Kreb Cycle

Part 2: The Kreb Cycle Reactions

Welcome to part two of our lecture series on the Kreb Cycle. This tutorial will go through the metabolic reactions of the Kreb Cycle in more detail.

Kreb Cycle Overview Image from Narayanese, WikiUserPedia, YassineMrabet, TotoBaggins The Kreb Cycle has a total of 8 metabolic reactions involved in the full oxidation of our food molecules into carbon dioxide.

The Kreb Cycle Shown Enzymatically Citrate Synthase Image modified from : Goodsell, D. (2012) Molecule of the Month, Protein Database Aconitase Malate Dehydrogenase Isocitrate Dehydrogenase a-ketoglutarate Dehydrogenase Succinyl-CoA Synthetase Succinate Dehydrogenase Fumarase The citric acid cycle, also known as the Krebs cycle or the tricarboxylic acid cycle, is at the center of cellular metabolism, playing a starring role in both the process of energy production and biosynthesis. It finishes the sugar-breaking job started in glycolysis and fuels the production of ATP in the process. It is also a central hub in biosynthetic reactions, providing intermediates that are used to build amino acids and other molecules. The citric acid cycle enzymes, shown here, are found in all cells that use oxygen, and even in some cells that don’t. In addition, all of these enzymatic reactions take place in the matrix of the mitochondria, where they are tethered closely to the innermembrane, and in fact, the succinate dehydrogenase is actually a membrane bound protein and participates in the next stage of the process, the electron transport chain.

Reaction 1: Citrate Synthase

Oxaloacetate + Acetyl-CoA Citrate

Image from : Hbf Citrate Synthase The first reaction in the citric acid cycle is the formation of citric acid (or citrate) from acetal-coA and oxaloacetate. CoASH is released in the process. Note that the two carboxylic acid groups shown in blue originate from oxaloacetate, while the acetate-derived carboxylic acid group and methyl carbon are shown in red. This will allow you to visualize what happens to these positions throughout the Kreb cycle reactions.

Reaction 3: Isocitrate Dehydrogenase

• Dehydrogenase AND Decarboxylase Activity Isocitrate Dehydrogenase

Isocitrate a-Ketoglutarate

Within the citric acid cycle, isocitrate, produced from the isomerization of citrate, undergoes both oxidation and decarboxylation to form alpha-Ketoglutarate. CO2 is released and a molecule of NADH is formed in the process.

Isocitrate Dehydrogenase Image from : Goodsell, D. (2012) Molecule of the Month, Protein Database Reaction Mechanism involves NAD+^ and Mg2+^ as cofactors This is accomplished by the isocitrate dehydrogenase enzyme. Note that the enzyme is named for its oxidation role, not the decarboxylation step. Isocitrate dehydrogenase uses NAD+ and Mg2+ as cofactors within the reaction mechanism that is shown on the next few slides.

Isocitrate Dehydrogenase Image from : Haynathart Step 2: Decarboxylase & Lyase Activity In step 2, the decarboxylation occurs. In this reaction mechanism, the Mg2+ cofactor stabilizes the carbonyl function group and the C1 carboxylic acid functional group. An acidic base within the active site drives the decarboxylation which forms an alkene intermediate. Again, the carbonyl oxygen is charge stabilized by interaction with the metal cofactor.

Isocitrate Dehydrogenase Image from : Haynathart Step 3: Ketone Formation As the carbonyl electrons fall back into the molecule forming the ketone functional group, tthe pi-bond between the C2 and C3 positions, abstracts a proton from an acidic residue in the active site to form alpha-ketoglutarate. Take a moment to compare this mechanism with that found in the Pyruvate Dehydrogenase Complex, and that of the Glyceraldehyde 3- Phosphate Dehydrogenase from the Glycolytic pathway. This exemplifies the many different ways dehydrogenase enzymes can work to oxidize biological molecules.

a-Ketoglutarate Dehydrogenase Figure Modified from Stuart, et al (2014) Cancer and Metabolism 2: E E H 2 O (ETC) glutamate TPP Lipoamide E CoASH Succinyl-CoA SH HS FAD FADH 2 Similar to the pyruvate dehydrogenase (PDH), the alpha-ketoglutarate dehydrogenase (KGDH) also uses CoASH as a substate, and TPP, Lipoamide, FAD, and NAD+ as cofactors. In this case TPP attacks the alpha ketoglutarate C2 carbonyl carbon, mediating the release of the CO2 that was originally from the acetate molecule. The remaining succinyl group is then transferred to the lipoamide cofactor. Partial reduction of the lipoamide from the alpha-ketoglutarate has also occurred at this step. On the E2 complex, the succinyl group is transferred to Coenzyme-A as a thioester and released from the enzyme. The lipoamide cofactor is fully reduced in the process. The lipoamide cofactor releases the electrons the bound FAD housed on the E3 subunit. This restores the oxidized state of lipoamide for a second round of enzymatic function. Finally, the electrons are moved from the FADH2 to the labile NAD+ cofactor where they can be transported to the electron transport chain.

Reaction 5: Succinyl-CoA Synthetase

• Substrate-Level Phosphorylation Succinyl-CoA Synthetase

Succinyl-CoA Succinate

The Succinyl-CoA synthetase is an enzyme that creates a molecule of GTP (ATP equivalent) through the phosphorylation of GDP. This process releases the Coenzyme A and forms a molecule of succinate.

Succinate Dehydrogenase Image modified from : Goodsell, D. (2012) Molecule of the Month, Protein Database Matrix Intermembrane Space This step is performed by a protein complex that is bound in the membrane of the mitochondrion. It links this citric acid cycle task directly to the electron transport chain (ETC) and makes the transfer of electrons harvested from the food molecules streamlined. It first extracts hydrogen atoms from succinate, transferring them to the carrier FAD. The resulting product is fumarate. Interestingly, this protein is also Complex II in the electron transport chain, where it can directly transfer the electrons harvested from the succinate into the ETC. We will come back to this complex in the next section covering the ETC.

Reaction 7: Fumarase

• Hydrolase Fumarase

Fumarate Malate

In reaction 7, the lyase, known as fumarase, converts fumarate to malate. This enzyme is also a hydrolase, as water is incorporated into the final structure (shown in pink, here).

Energy Yield in Kreb Cycle

• Per Cycle
  • 3 NADH/H+
  • 1 FADH 2
  • 1 GTP (ATP equivalents)
• Per Glucose = 2 Cycles (2 Acetyl-CoA molecules)
  • 6 NADH/H+
  • 2 FADH 2
  • 2 GTP (ATP equivalents) Overall the total energy yielded in one turn of the citric acid cycle are 3 molecules of NADH & H+, 1 FADH2, and 1 GTP. This is doubled for the energy potential in 1 glucose molecule as two molecules of acetyl-CoA will enter the Kreb cycle.

Regulation of the Kreb Cycle

Three Major Regulatory Steps

• Pyruvate Dehydrogenase – The Gatekeeper of the Kreb

Cycle

• Isocitrate Dehydrogenase
• a-Ketoglutarate Dehydrogenase Regulation of this pathways is mediated primarily by three of the major dehydrogenase enzymes in the pathway. These include: Pyruvate dehydrogenase which serves as a gatekeeper prior to the beginning of the Kreb Cycle, followed by the next two dehydrogenase/carboxylase steps in the pathway, mediated by isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase. Essentially all of the steps were CO2 is released. All of these enzymes are highly regulated by the energy load of the cell.