Understanding metabolism and Krebs cycle regulation

For any living cell or organism to function, there are some chemical reactions that are required to take place in the body. These reactions are referred to as metabolism. Over 700 years ago, metabolic studies began in Damascus, Syria by Ibn al-Nafis, an Arab scholar and anatomist in 1260, who also discovered the pulmonary movement of blood (Iskandar). The study grew after Hans Krebs’ discovery of the Citric Acid cycle in 1937 providing a broader understanding of metabolism.

Metabolism

Metabolism is derived from ‘metabolismos’, Greek word for ‘change’. Ibn Nafis, in his work ‘The treatise of Kamil on the Prophet’s Biography’ said ‘both the body and its parts are in a continuous state of dissolution and nourishment, so they are inevitably undergoing permanent change’ (Al-Roubi, 1982). It is these changes that helps in maintaining the living state of organisms and it comprises of catabolism and anabolism which means breaking down and building up respectively. For metabolism to occur, energy is required. The energy is usually in form Adenosine Triphosphate, ATP and it is often referred to as the energy currency of the cell. This energy is either being used up or released in a metabolic reaction. Catabolic reactions release energy as heat when it breaks down large molecules into simpler ones while anabolic reactions uses energy for the synthesis of complex biomolecules from smaller precursors.

The biochemical reactions in metabolism are called biochemical pathways which are regulated by (mostly) specific enzymes. Regulation of these enzymes in turn regulates the metabolic processes thereby controlling the conversion of substrates to products. The end products of metabolic processes (fatty acids, carbohydrate and are often converted to acetyl CoA which is used by the TCA cycle for energy production.

The Krebs cycle

British biochemist, Hans Krebs discovered the Krebs cycle in 1937. The Krebs cycle (also known as the Citric Acid or Tricarboxylic Acid (TCA) cycle) is the process through which aerobic cellular metabolism occurs i.e the body uses glycolysis to produce energy. Hans Krebs after who the cycle is named received the 1953 Nobel Prize in Medicine for his ‘discovery’. Krebs cycle is the hub of cellular metabolism involved in both degradation and building up process. Here, the carbon atoms within the acetyl group from glycolysis are oxidized for energy (ATP) production.

This cycle involves a series of reactions involving:

1. A substrate, oxaloacetate, which is modified in every reaction.
2. Acetyl–CoA, from which energy is extracted.
3. Energy transport reactants, which collect the extracted energy, and
4. The controlling enzymes, which regulate the steps of the cycle.

This cycle is ubiquitous in living organisms, single and multi-celled, both plants and animals – including humans. Organizationally, the process is often divided into 8 steps, one for each controlling enzyme, usually beginning with the combination of the Oxaloacetate substrate to the Acetyl–CoA, which is produced from either glycolysis or pyruvate oxidation.

The Krebs cycle is the primary metabolic pathway through which aerobic energy is released from carbohydrates, proteins, and fats in a useable form. When measuring the energy production of the Krebs cycle, the output is measured in molecules of ATP (Adenosine triphosphate) per molecule of glucose. In total, the theoretical yield of cellular respiration (including the Krebs cycle) from one molecule of glucose is 38 molecules of ATP, but in practice, the actual yield is closer to 30-32 ATP. Since one molecule of glucose produces two molecules of Acetyl–CoA, the Krebs cycle’s energy output is usually expressed as the product of the two cycles necessary to break down both Acetyl–CoA. Two Krebs cycles create two GTP, Guanosine triphosphate, which can be readily converted into 2 ATP. The other energy-producing products of the Krebs cycle (NADH, and QH2) theoretically generate an additional 22 ATP, but in practice produce closer to18 ATP via the mitochondrial electron transport chain. This practical difference results from energy lost from the active transport of various reactants as well as the leakage of electrons within the electron transport chain.

Steps in the Krebs cycle

The Krebs cycle releases energy from Acetyl–CoA, but the cellular challenge is to release the energy gradually and in useable forms. So the pathway:

1. Links the Acetyl-CoA’s acetyl group (2-carbon) to the substrate (4-carbon) to make a 6-carbon molecule;
2. Rearranges the 6-carbon molecule to a more reactive form;
3. Removes one of the substrate’s carbon molecules to form a 5-carbon molecule and release energy
4. Removes another of the substrate’s carbon atoms, to form a 4- carbon molecule, and release energy; and
5. Rearranges the 4-carbon molecule several times to re-create first substrate, releasing energy in the process. The key observation is that the substrate is first manipulated and its carbon atoms are released in the form of CO2 and only then are the atoms in the acetate (from the Acetyl–CoA) rearranged to re-create the substrate.

The Krebs cycle pathway

Source: Citric_acid_cycle_noi.svg

Step 1: Citrate synthase

The first step is to put energy into the system. The Citrate synthase links to the Oxaloacetate substrate which can then bind to the Acetyl–CoA’s acetyl group, which then releases the Co-enzyme A. This produces the very familiar and common citric acid. It is this six-carbon molecule that will be broken down, and remolded back into Oxaloacetate.

Step 2: Aconitase

Unfortunately, citrate is too stable for the reactions that follow. So, the Acontiase links to the Citrate to move one of its oxygen atoms to create a more unstable citrate isomer. It does this by extracting a water molecule producing cis– Aconitate, and then reattaching the water to produce D–Isocitrate.

Step 3: Isocitrate dehydrogenase

With the citrate rearranged, the process begins in earnest; the Isocitrate dehydrogenase links to the D–Isocitrate, which transfers an electron to the NAD+, Nicotinamide adenine dinucleotide, producing its energized form NADH. With the electron removed, the enzyme then detaches a carbon atom to form a molecule of carbon dioxide. This transforms the substrate from a 6- carbon molecule to a 5-carbon molecule.

Step 4: α-Ketoglutarate dehydrogenase

This step involves a highly-developed complex of 24 enzymes. Labelled here α–Ketoglutarate dehydrogenase, this complex transfers also electrons to NAD + producing NADH, removes another carbon atom as carbon dioxide (transforming the substrate from a 5-carbon to a 4-carbon molecule), and relinks the Coenzyme A to the substrate.

Step 5: Succinyl-CoA synthetase

This step directly produces ATP because the substrate’s link to Coenzyme A is sufficiently energetic to power the reaction. In mitochondria, the enzyme links to the Succinyl–CoA and uses the energy from releasing the coenzyme, to add a phosphateto GDP to produce GTP. In the cytoplasm, a variation on this enzyme can produce ATP directly. This also begins the manipulation of the substrate to re-produce its original form.

Step 6: Succinate dehydrogenase

With the carbons removed, the rearrangement process begins manipulating the hydrogen. When the Succinate dehydrogenase links to the substrate, it releases two hydrogen atoms attaching them to a carrier, ubiquinone (Q), or FAD Flavinadenine dinucleotide. With the additional 2 electrons, ubiquinone forms ubiquinol (QH 2 or FADH 2) which is then transferred to power the electron transport chain.

Step 7: Fumarase

Fumarase continues the rearrangement process by adding Hydrogen and Oxygen back into the substrate that had been previously removed.

Step 8: Malate dehydrogenase

Finally, the Malate dehydrogenase recreates the Oxaloacetate substrate and moves electrons from the NAD +to form NADH, the last energy produced by the Krebs cycle. Interestingly, this Malate–Oxaloacetate reaction is also used to move anaerobic energy from the cytoplasm into the mitochondria. While anaerobic reactions produce NADH, it cannot move from the cytoplasm to the mitochondria to be processed in the electron transport chain, but the Malate can be transported across the mitochondria’s membrane, so the anaerobic NADH transforms Oxaloacetate into Malate, which is then converted back into Oxaloacetate to produce NADH for the production of ATP.

Regulation of the Krebs cycle

The regulation of key enzymes in metabolic pathways, by allosteric effectors and by covalent modification, ensures the production of intermediates at the rates required to keep the cell in a stable steady state while avoiding wasteful overproduction. The flow of carbon atoms from pyruvate into and through the citric acid cycle is under tight regulation at two levels: the conversion of pyruvate to acetyl-CoA, the starting material for the cycle (the pyruvate dehydrogenase complex reaction), and the entry of acetyl-CoA into the cycle (the citrate synthase reaction).

1. Allosteric regulation by metabolites. The regulation of the citric acid cycle is largely determined by product inhibition and substrate availability. If the cycle were permitted to run unchecked, large amounts of metabolic energy could be wasted in overproduction of reduced coenzyme such as NADH and ATP. The major substrate of the cycle is ADP which gets converted to ATP. A reduced amount of ADP causes accumulation of precursor NADH which in turn can inhibit a number of enzymes. NADH, a product of all dehydrogenases in the citric acid cycle with the exception of succinate dehydrogenase, inhibits pyruvate dehydrogenase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and also citrate synthase. Acetyl-coA inhibits pyruvate dehydrogenase, while succinyl-CoA inhibits alpha-ketoglutarate dehydrogenase and citrate synthase. When tested in-vitro with TCA enzymes, ATP inhibits citrate synthase and α-ketoglutarate dehydrogenase; however, ATP levels do not change more than 10% in vivo between rest and vigorous exercise. There is no known allosteric mechanism that can account for large changes in reaction rate from an allosteric effector whose concentration changes less than 10%.
2. Citrate is used for feedback inhibition, as it inhibits phosphofructokinase, an enzyme involved in glycolysis that catalyzes the formation of fructose 1, 6-bisphosphate , a precursor of pyruvate. This prevents a constant high rate of flux when there is an accumulation of citrate and a decrease in substrate for the enzyme.
3. Regulation by calcium. Calcium is also used as a regulator in the citric acid cycle. Calcium levels in the mitochondrial matrix can reach up to the tens of micromolar levels during cellular activation. It activates pyruvate dehydrogenase phosphatase which in turn activates the pyruvate dehydrogenase complex. Calcium also activates isocitrate dehydrogenase and α-ketoglutarate dehydrogenase. This increases the reaction rate of many of the steps in the cycle, and therefore increases flux throughout the pathway.
4. Transcriptional regulation: Recent work has demonstrated an important link between intermediates of the citric acid cycle and the regulation of hypoxia-inducible factors ( HIF ). HIF plays a role in the regulation of oxygen homeostasis, and is a transcription factor that targets angiogenesis, vascular remodeling, glucose utilization, iron transport and apoptosis. HIF is synthesized constitutively, and hydroxylation of at least one of two critical proline residues mediates their interaction with the Von Hippel-Lindau E3 ubiquitin ligase complex, which targets them for rapid degradation. This reaction is catalyzed by prolyl 4-hydroxylases. Fumarate and succinate have been identified as potent inhibitors of prolyl hydroxylases, thus leading to the stabilization of HIF.

Regulation of metabolism is important to prevent wastage of energy and metabolites, to control metabolic flux as well as to maintain homeostasis. When a metabolic pathway is not regulated, substances are either produced in excess or broken down to a point of depletion where it becomes unavailable for the body’s normal functioning.

References

Lane, Nick (2009). Life Ascending: The Ten Great Inventions of Evolution. New York: W. W. Norton & Co.

ISBN 978-0-393-06596-1.

Chinopoulos C (2013). “Which way does the citric acid cycle turn during hypoxia? The critical role of α-ketoglutarate dehydrogenase complex”. Journal of Neuroscience Research. 91: 1030–43.

Voet D, Voet JG (2004). Biochemistry (3rd Ed.). New York: John Wiley & Sons, Inc. p. 615.