Principles of Biochemistry/Krebs cycle or Citric acid cycle

The citric acid cycle — also known as the tricarboxylic acid cycle (TCA cycle), the Krebs cycle, or the Szent-Györgyi-Krebs cycle — is a series of enzyme-catalysed chemical reactions, which is of central importance in all living cells that use oxygen as part of cellular respiration. In eukaryotic cells, the citric acid cycle occurs in the matrix of the mitochondrion. The components and reactions of the citric acid cycle were established by seminal work from Albert Szent-Györgyi and Hans Krebs. In aerobic organisms, the maerianne is part of a metabolic pathway involved in the chemical conversion of carbohydrates, fats and proteins into carbon dioxide and water to generate a form of usable energy. Other relevant reactions in the pathway include those in glycolysis and pyruvate oxidation before the citric acid cycle, and oxidative phosphorylation after it. In addition, it provides precursors for many compounds including some amino acids and is therefore functional even in cells performing fermentation.

Overview of the citric acid cycle

Enzymes in Citric acid cycle pathway

Citrate synthase

The enzyme citrate synthase (E.C. 2.3.3.1 [previously 4.1.3.7]) exists in nearly all living cells and stands as a pace-making enzyme in the first step of the Citric Acid Cycle (or Krebs Cycle). Citrate synthase is localized within eukaryotic cells in the mitochondrial matrix, but is encoded by nuclear DNA rather than mitochondrial. It is synthesized using cytoplasmic ribosomes, then transported into the mitochondrial matrix. Citrate synthase is commonly used as a quantitative enzyme marker for the presence of intact mitochondria. Citrate synthase catalyzes the condensation reaction of the two-carbon acetate residue from acetyl coenzyme A and a molecule of four-carbon oxaloacetate to form the six-carbon citrate.Oxaloacetate will be regenerated after the completion of one round of the Krebs Cycle.

• 
  acetyl-CoA
• 
  Oxaloacetic acid
• 
  Citric acid

acetyl-CoA + oxaloacetate + H₂O → citrate + CoA-SH

Oxaloacetate is the first substrate to bind to the enzyme. This induces the enzyme to change its conformation, and creates a binding site for the acetyl-CoA. Only when this citroyl-CoA has formed will another conformational change cause thioester hydrolysis and release coenzyme A. This ensures that the energy released from the thioester bond cleavage will drive the condensation.

Catalytic mechanism of the breakdown of isocitrate into oxalosuccinate, then into a final product of alpha-ketuglutarate. The oxalosuccinate intermediate is hypothetical; it has never been observed in the decarboxylating version of the enzyme.

Aconitase

Aconitase (aconitate hydratase; EC 4.2.1.3) is an enzyme that catalyses the stereo-specific isomerization of citrate to isocitrate via cis-aconitate in the tricarboxylic acid cycle, a non-redox-active process.

• 
  Citric acid
• 
  Aconitic acid
• 
  Isocitric acid

Isocitrate dehydrogenase

Isocitrate dehydrogenase (EC 1.1.1.42) and (EC 1.1.1.41), also known as IDH, is an enzyme that participates in the citric acid cycle. It catalyzes the third step of the cycle: the oxidative decarboxylation of isocitrate, producing alpha-ketoglutarate (α-ketoglutarate) and CO2 while converting NAD+ to NADH. This is a two-step process, which involves oxidation of isocitrate (a secondary alcohol) to oxalosuccinate (a ketone), followed by the decarboxylation of the carboxyl group beta to the ketone, forming alpha-ketoglutarate. Another isoform of the enzyme catalyzes the same reaction, however this reaction is unrelated to the citric acid cycle, is carried out in the cytosol as well as the mitochondrion and peroxisome and uses NADP+ as a cofactor instead of NAD+.

Within the citric acid cycle, isocitrate, produced from the isomerization of citrate, undergoes both oxidation and decarboxylation. Using the enzyme Isocitrate Dehydrogenase (IDH), isocitrate is held within its active site by surrounding arginine, tyrosine, asparagine, serine, threonine, and aspartic acid amino acids. The first box shows the overall isocitrate dehydrogenase reaction. The reactants necessary for this enzyme mechanism to work are isocitrate, NAD+/NADP+, and Mn2+ or Mg2+. The products of the reaction are alpha-ketoglutarate, carbon dioxide, and NADH + H+/NADPH + H+. Water molecules are used to help deprotonate the oxygens (O3) of isocitrate. The second box is Step 1, which is the oxidation of the alpha-C (C#2).Oxidation is the first step that isocitrate goes through. In this process, the alcohol group off the alpha-carbon (C#2) is deprotonated and the electrons flow to the alpha-C forming a ketone group and removing a hydride off C#2 using NAD+/NADP+ as an electron accepting cofactor. The oxidation of the alpha-C allows for a position where electrons (in the next step) will be coming down from the carboxyl group and pushing the electrons (making the double bonded oxygen) back up on the oxygen or grabbing a nearby proton off a nearby Lysine amino acid. The third box is Step 2, which is the decarboxylation of oxalosuccinate. In this step, the carboxyl group oxygen is deprotonated by a nearby Tyrosine amino acid and those electrons flow down to carbon 2. Carbon dioxide leaves the beta carbon of isocitrate as a leaving group with the electrons flowing to the ketone oxygen off the alpha-C placing a negative charge on the oxygen of the alpha-C and forming an alpha-beta unsaturated double bond between carbons 2 and 3. The lone pair on the alpha-C oxygen picks up a proton from a nearby Lysine amino acid. The fourth box is Step 3, which is the saturation of the alpha-beta unsaturated double bond between carbons 2 and 3. In this step of the reaction,[5][6] Lysine deprotonates the oxygen off the alpha carbon and the lone pair of electrons on the oxygen of the alpha carbon comes down reforming the ketone double bond and pushing the lone pair (forming the double bond between the alpha and beta carbon) off, picking up a proton from the nearby Tyrosine amino acid. This reaction results in the formation of alpha-ketoglutarate, NADH + H+/NADPH + H+, and CO2.

α-ketoglutarate dehydrogenase

The oxoglutarate dehydrogenase complex (OGDC) or α-ketoglutarate dehydrogenase complex is an enzyme complex, most commonly known for its role in the citric acid cycle. The reaction catalyzed by this enzyme in the citric acid cycle is:

α-ketoglutarate + NAD⁺ + CoA → Succinyl CoA + CO₂ + NADH

This reaction proceeds in three steps: decarboxylation of α-ketoglutarate, reduction of NAD+ to NADH, and subsequent transfer to CoA, which forms the end product, succinyl CoA. ΔG°' for this reaction is -7.2 kcal mol-1. The energy needed for this oxidation is conserved in the formation of a thioester bond of succinyl CoA.

Succinyl coenzyme A synthetase

Succinyl coenzyme A synthetase (succinate thiokinase) catalyzes the formation of succinate and coenzyme-A, a 4-carbon metabolite, from succinyl-CoA. Succinyl-CoA synthetase catalyzes a reversible step in the citric acid cycle, which involves the substrate-level phosphorylation of GDP.

• 
  succinyl-CoA
• 
  succinate

Succinate dehydrogenase

Succinate dehydrogenase or succinate-coenzyme Q reductase (SQR) or Complex II is an enzyme complex, bound to the inner mitochondrial membrane of mammalian mitochondria and many bacterial cells. It is the only enzyme that participates in both the citric acid cycle and the electron transport chain.

In step 8 of the citric acid cycle, SQR catalyzes the oxidation of succinate to fumarate with the reduction of ubiquinone to ubiquinol. This occurs in the inner mitochondrial membrane by coupling the two reactions together.

Fumarase

Fumarase (or fumarate hydratase) is an enzyme that catalyzes the reversible hydration/dehydration of Fumarate to S-malate. Fumarase comes in two forms: mitochondrial and cytosolic. The mitochondrial isoenzyme is involved in the Krebs Cycle (also known as the Citric Acid Cycle), and the cytosolic isoenzyme is involved in the metabolism of amino acids and fumarate. Subcellular localization is established by the presence of a signal sequence on the amino terminus in the mitochondrial form, while subcellular localization in the cytosolic form is established by the absence of the signal sequence found in the mitochondrial variety. This enzyme participates in two other metabolic pathways: reductive carboxylation cycle (CO2 fixation) and in renal cell carcinoma also.

Malate dehydrogenase

Malate dehydrogenase (EC 1.1.1.37) (MDH) is an enzyme in the citric acid cycle that catalyzes the conversion of malate into oxaloacetate (using NAD+) and vice versa (this is a reversible reaction). Malate dehydrogenase is not to be confused with malic enzyme, which catalyzes the conversion of malate to pyruvate, producing NADPH. Malate dehydrogenase is also involved in gluconeogenesis, the synthesis of glucose from smaller molecules. Pyruvate in the mitochondria is acted upon by pyruvate carboxylase to form oxaloacetate, a citric acid cycle intermediate. In order to get the oxaloacetate out of the mitochondria, malate dehydrogenase reduces it to malate, and it then traverses the inner mitochondrial membrane. Once in the cytosol, the malate is oxidized back to oxaloacetate by cytosolic malate dehydrogenase. Finally, phosphoenol-pyruvate carboxy kinase (PEPCK) converts oxaloacetate to phosphoenol pyruvate.

Steps of Krebs cycle or Citric acid cycle pathway

Two carbon atoms are oxidized to CO₂, the energy from these reactions being transferred to other metabolic processes by GTP (or ATP), and as electrons in NADH and QH₂. The NADH generated in the TCA cycle may later donate its electrons in oxidative phosphorylation to drive ATP synthesis; FADH₂ is covalently attached to succinate dehydrogenase, an enzyme functioning both in the TCA cycle and the mitochondrial electron transport chain in oxidative phosphorylation. FADH₂, therefore, facilitates transfer of electrons to coenzyme Q, which is the final electron acceptor of the reaction catalyzed by the Succinate:ubiquinone oxidoreductase complex, also acting as an intermediate in the electron transport chain.

The citric acid cycle is continuously supplied with new carbon in the form of acetyl-CoA, entering at step 1 below.

	Substrates	Products	Enzyme	Reaction type	Comment
1	Oxaloacetate + Acetyl CoA + H2O	Citrate + CoA-SH	Citrate synthase	Aldol condensation	rate-limiting stage (irreversible), extends the 4C oxaloacetate to a 6C molecule
2	Citrate	cis-Aconitate + H2O	Aconitase	Dehydration	reversible isomerisation
3	cis-Aconitate + H2O	Isocitrate		Hydration
4	Isocitrate + NAD+	Oxalosuccinate + NADH + H +	Isocitrate dehydrogenase	Oxidation	generates NADH (equivalent of 2.5 ATP)
5	Oxalosuccinate	α-Ketoglutarate + CO2		Decarboxylation	irreversible stage, generates a 5C molecule
6	α-Ketoglutarate + NAD+ + CoA-SH	Succinyl-CoA + NADH + H+ + CO2	α-Ketoglutarate dehydrogenase	Oxidative decarboxylation	irreversible stage, generates NADH (equivalent of 2.5 ATP), regenerates the 4C chain (CoA excluded)
7	Succinyl-CoA + GDP + Pi	Succinate + CoA-SH + GTP	Succinyl-CoA synthetase	substrate-level phosphorylation	or ADP→ATP instead of GDP→GTP, generates 1 ATP or equivalent
8	Succinate + ubiquinone (Q)	Fumarate + ubiquinol (QH2)	Succinate dehydrogenase	Oxidation	uses FAD as a prosthetic group (FAD→FADH2 in the first step of the reaction) in the enzyme, generates the equivalent of 1.5 ATP
9	Fumarate + H2O	L-Malate	Fumarase	H2O addition (hydration)
10	L-Malate + NAD+	Oxaloacetate + NADH + H+	Malate dehydrogenase	Oxidation	reversible (in fact, equilibrium favors malate), generates NADH (equivalent of 2.5 ATP)

Mitochondria in animals, including humans, possess two succinyl-CoA synthetases: one that produces GTP from GDP, and another that produces ATP from ADP. Plants have the type that produces ATP (ADP-forming succinyl-CoA synthetase). Several of the enzymes in the cycle may be loosely-associated in a multienzyme protein complex within the mitochondrial matrix.

The GTP that is formed by GDP-forming succinyl-CoA synthetase may be utilized by nucleoside-diphosphate kinase to form ATP (the catalyzed reaction is GTP + ADP → GDP + ATP).