Krebs cycle

Learning scheme of the citric acid cycle.

The Krebs cycle (citric acid cycle or tricarboxylic acid cycle) is a metabolic pathway, that is, a succession of chemical reactions, which is part of cellular respiration in all aerobic cells, where stored energy is released through the oxidation of acetyl-CoA derived from carbohydrates, lipids and proteins into carbon dioxide and chemical energy in the form of ATP. In the eukaryotic cell, the Krebs cycle takes place in the mitochondrial matrix.

In addition, the cycle provides precursors of certain amino acids, as well as the reducing agent NADH that is used in numerous biochemical reactions. Its central importance to many biochemical pathways suggests that it is one of the first established components of cellular metabolism and points to an abiogenic origin.

In aerobic organisms, the Krebs cycle is part of the catabolic pathway that carries out the oxidation of carbohydrates, fatty acids and amino acids to produce CO2, releasing usable energy: reducing power and GTP (ATP is produced in some microorganisms).

The oxidative metabolism of carbohydrates, lipids, and proteins is often divided into three stages, of which the Krebs cycle is the second. In the first stage, the carbons of these macromolecules give rise to acetyl-CoA, and includes amino acid catabolic pathways (eg, oxidative deamination), beta-oxidation of fatty acids, and glycolysis. The third stage is oxidative phosphorylation, in which the reducing power (NADH and FADH₂) generated is used for ATP synthesis according to the chemiosmotic coupling theory.

The Krebs cycle also provides precursors for many biomolecules, such as certain amino acids. For this reason it is considered an amphibolic pathway, that is, catabolic and anabolic at the same time.

The name of this metabolic pathway is derived from citric acid (a type of tricarboxylic acid) being consumed and then regenerated by this sequence of reactions to complete the cycle, or also known as the Krebs cycle as it was discovered by the German Hans Adolf Krebs, who won the Nobel Prize in Physiology or Medicine in 1953, together with Fritz Lipmann.

Many of the components and reactions of the citric acid cycle were established in the 1930s by the research of Nobel laureate Albert Szent-Györgyi, for which he received the Nobel Prize in 1937, specifically for his discoveries related to citric acid fumaric, a key component of this metabolic pathway. The citric acid cycle was finally identified in 1937 by Hans Adolf Krebs, at the University of Sheffield, for which he received the Nobel Prize in Medicine in 1953.

Evolution

The components of the cycle were derived from anaerobic bacteria, and the cycle itself has possibly evolved more than once. Theoretically, there are several alternatives to the citric acid cycle, however this cycle appears to be the most efficient. If various alternatives to the Krebs cycle had evolved independently, they all seem to have converged on this path.

Overview

The citric acid cycle is a key metabolic pathway that unifies the metabolism of carbohydrates, fats, and proteins. The reactions of the cycle are carried out by 8 enzymes that completely oxidize the acetate, in the form of acetyl-CoA, and two molecules of carbon dioxide and water are released for each one. Through the catabolism of sugars, fats, and proteins, a two-carbon organic product acetate is produced in the form of acetyl-CoA which enters the citric acid cycle. The reactions of the cycle also convert three equivalents of nicotinamide adenine dinucleotide (NAD+) to three equivalents of reduced NAD+ (NADH), one equivalent of flavin adenine dinucleotide (FAD) to one of FADH2, and one equivalent of guanosine diphosphate) Y inorganic phosphate (Pi) into a guanosine triphosphate (GTP). The NADH and FADH2 generated by the citric acid cycle are in turn used by the oxidative phosphorylation pathway to generate energy-rich adenosine triphosphate (ATP).

One of the primary sources of acetyl-CoA is the breakdown of sugars by glycolysis to produce pyruvate which in turn is decarboxylated by the enzyme pyruvate dehydrogenase to generate acetyl-CoA.

Acetil CoA

The product of this reaction, acetyl-CoA, is the starting point for the citric acid cycle.

The citric acid cycle begins with the transfer of a two-carbon acetyl group from acetyl-CoA to the four-carbon acceptor compound (oxaloacetate) to form a six-carbon compound (citrate).

The citrate then goes through a series of chemical transformations, losing two carboxyl groups as CO₂. The carbons lost as CO₂ originate from what was oxaloacetate, not directly from acetyl-CoA. The carbons donated by acetyl-CoA become part of the oxaloacetate carbon backbone after the first round of the citric acid cycle. Loss of the acetyl-CoA donated carbons as CO₂ requires several turns of the citric acid cycle. However, due to the role of the citric acid cycle in anabolism, they may not be lost, as many TCA cycle intermediates are also used as precursors for the biosynthesis of other molecules.

Most of the energy available from the oxidative steps of the cycle is transferred as energy-rich electrons to NAD +, forming NADH. For each acetyl group that enters the citric acid cycle, three NADH molecules are produced.

Electrons are also transferred to the electron acceptor Q, forming QH2.

At the end of each cycle, the four-carbon oxaloacetate has been regenerated, and the cycle continues.

Krebs Cycle Reactions

The Krebs cycle takes place in the mitochondrial matrix in the eukaryotic cell.

Krebs Cycle in the mitochondrial matrix.

Acetyl-CoA (Acetyl Coenzyme A) is the main precursor of the cycle. Citric acid (6 carbons) or citrate is obtained in each cycle by condensation of an acetyl-CoA (2 carbons) with a molecule of oxaloacetate (4 carbons). Citrate produces one molecule of oxaloacetate and two CO₂ in each cycle, so the net balance of the cycle is:

The two carbons of acetyl-CoA are oxidized to CO₂, and the energy it had accumulated is released in the form of chemical energy: GTP and reducing power (high potential electrons): NADH and FADH2. NADH and FADH₂ are coenzymes (enzyme-binding molecules) capable of accumulating energy in the form of reducing power for its conversion into chemical energy in oxidative phosphorylation.

The FADH₂ of succinate dehydrogenase (complex II of the electron transport chain), unable to be released from the enzyme, must be oxidized again in situ. FADH₂ donates its two hydrogens to ubiquinone (coenzyme Q), which is reduced to ubiquinol (QH₂) and leaves the enzyme.

The reactions are:

Simplified vision and process performance

• The final step is the oxidation of the Krebs cycle, producing an oxaloacetate and two CO2.
• Acetyl-CoA reacts with an oxaloacetate molecule (4 carbons) to form citrate (6 carbons), by means of a condensation reaction.
• Through a series of reactions, the citrate becomes oxaloacetate again.
• During these reactions, 2 carbon atoms of the citrate (6C) are substrated to give oxalacetate (4C); such carbon atoms are released as CO₂
• The cycle consumes net 1 acetyl-CoA and produces 2 CO₂. It also consumes 3 NAD⁺ and 1 FAD, producing 3 NADH + 3 H⁺ and 1 FADH₂.
• The performance of a cycle is (for each pyruvate molecule): 1 GTP, 3 NADH +3H⁺, 1 FADH₂, 2CO₂.
• Each NADH, when oxide in the respiratory chain, will originate 3 ATP molecules (3 x 3 = 9), while FADH₂ will give rise to 2 ATP. Therefore, 9 + 2 + 1 GTP = 12 ATP per acetyl-CoA that enters the Krebs cycle.
• Each glucose molecule produces (via glucolysis) two piruvate molecules, which in turn produce two acetyl-COA, so for each glucose molecule in the Krebs cycle it occurs: 4CO₂, 2 GTP, 6 NADH + 6H ⁺, 2 FADH₂; total 24 ATP.

Regulation

Many of the enzymes of the Krebs cycle are regulated by negative feedback (feedback), by allosteric binding of ATP, which is a product of the pathway and an indicator of the energy level of the cell. These enzymes include the pyruvate dehydrogenase complex that synthesizes the acetyl-CoA required for the first reaction of the cycle from pyruvate (through an irreversible reaction), from glycolysis, or from glucogenic amino acid catabolism (i.e., the standard 20 amino acids except lysine and leucine). This enzyme is regulated by inhibition, product of NADH and acetyl-CoA, and covalent modification of the enzyme by phosphorylation. Also the enzymes citrate synthase, isocitrate dehydrogenase, and α-ketoglutaramate dehydrogenase, which catalyze the first three reactions of the Krebs cycle, They are inhibited by high concentrations of ATP. This regulation stops this degradative cycle when the energy level of the cell is good.

Some enzymes are also downregulated when the level of reducing power of the cell is high. The mechanism that takes place is a competitive inhibition by product (by NADH) of the enzymes that use NAD⁺ as substrate. Thus, among others, the pyruvate dehydrogenase and citrate synthase complexes are regulated.

Efficiency

The theoretical maximum yield of ATP through the oxidation of one glucose molecule in glycolysis, the citric acid cycle, and oxidative phosphorylation is thirty-eight (assuming three molar equivalents of ATP per NADH equivalent and two ATP per FADH ₂). In eukaryotes, two equivalents of NADH are generated in glycolysis, which occurs in the cytoplasm. Transport of these two equivalents into the mitochondria consumes two ATP equivalents, thus reducing net ATP production to thirty-six. Furthermore, inefficiencies in oxidative phosphorylation due to proton leakage across the mitochondrial membrane and ATP synthase/proton pump slippage normally reduces ATP production from NADH and FADH₂ below theoretical maximum yield. Observed yields are therefore closer to ~2.5 ATP per NADH and ~1.5 ATP per FADH₂, further reducing production net total ATP to approximately thirty. Evaluation of total ATP yield with recently revised ratios of protons to ATP provides an estimate of 29.85 ATP per glucose molecule.

Main pathways that converge in the Krebs cycle

The Krebs Cycle is a central metabolic pathway in which others converge, both anabolic and catabolic. They enter the cycle through different metabolites:

• Acetil-CoA:
  • Glucolysis
  • Oxidation of fatty acids
  • Production of collagen
• Malato:
  • Gluconeogenesis (by action of the MS or NADP-dependent dehydrogenase enzyme⁺; this enzyme converts pyruvate into malate using NADPH, CO₂ and H₂O).
• Oxalacetato:
  • Oxidation and biosynthesis of amino acids
• Fumarato:
  • Aspartate degradation, phenylanine and thyrosine
• Succinil-CoA
  • Biosynthesis of porphyrin
  • Degradation of isoleucin and methionine
  • Oxidation of fatty acids
• Alpha-cetoglutarato
  • Oxidation and biosynthesis of amino acids
• Citrate
  • Biosynthesis of fatty acids and cholesterol
• NADH and FADH
  • oxidative phosphorylation and electronic transport chain