KREB’S CYCLE

Submitted by

Dr. Sakshi Verma

Assistant Professor

Zoology Department

HMV, Jalandhar

kreb’s cycle

• The citric acid cycle—also known as the Krebs cycle, Szent–Györgyi–Krebs cycle or the TCA cycle —is a series of biochemical reactions to release the energy stored in nutrients through the oxidation of acetyl-CoA derived from carbohydrates, fats, and proteins. The chemical energy released is available under the form of ATP.
• The Krebs cycle is used by organisms that respire to generate energy, either by anaerobic respiration or aerobic respiration.
• In addition, the cycle provides precursors of certain amino acids, as well as the reducing agent NADH, that are used in numerous other reactions.

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kreb’s cycle

• Production of Acetyl-CoA (Activated Acetate): Pyruvate, formed after glycolysis, is oxidized to Acetyl-CoA and CO₂ .by the pyruvate dehydrogenase (PDH) complex, a cluster of enzymes—multiple copies of each of three enzymes—located in the mitochondria of eukaryotic cells and in the cytosol of prokaryotes. This reaction is called as an oxidative decarboxylation, an irreversible oxidation process in which the carboxyl group is removed from pyruvate as a molecule of CO₂ and the two remaining carbons become the acetyl group of acetyl-CoA

kreb’s cycle

• The PDH complex is a multienzyme complex in which a series of chemical intermediates remain bound to the enzyme molecules as a substrate is transformed into the final product. In this complex three different enzymes and Five cofactors, four derived from vitamins, participate in the reaction mechanism.
• The three enzymes of PDH complex are pyruvate dehydrogenase (E1), dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3)—each present in multiple copies.
• The five different coenzymes or prosthetic groups of PDH complex include thiamine pyrophosphate (TPP), flavin adenine dinucleotide (FAD), coenzyme A (CoA, sometimes denoted CoA-SH, nicotinamide adenine dinucleotide (NAD), and lipoate.
• Four different vitamins required in human nutrition are vital components of this system: thiamine (in TPP), riboflavin (in FAD), niacin (in NAD), and pantothenate (in CoA).

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kreb’s cycle

• Citric acid Cycle: the process by which acetyl- CoA undergoes oxidation is called as citric acid cycle.
• In eukaryotes, the entire set of reactions of the citric acid cycle takes place in mitochondria.
• In most prokaryotes, the enzymes of the citric acid cycle are in the cytosol, and the plasma membrane plays a role analogous to that of the inner mitochondrial membrane in ATP synthesis.
• The Citric Acid Cycle Has Eight Steps:

1. Condensation (Formation of Citrate): The first reaction of the cycle is the condensation of acetyl-CoA with oxaloacetate to form citrate, catalyzed by citrate synthase. The citrate has three carboxyl groups. Hence, the Kreb’s Cycle is also called as Tricarboxylic acid cycle or TCA cycle.

kreb’s cycle

• The Citric Acid Cycle Has Eight Steps:

2. Formation of Isocitrate via cis-Aconitate: Citrate undergoes reorganization in the presence of an enzyme aconitase, forming 6-carbon cis-aconitate and releasing water.

Then cis-aconitate is further reorganized into 6-carbon isocitrate by the enzyme, aconitase, with the addition of water.

kreb’s cycle

• The Citric Acid Cycle Has Eight Steps:

3. Oxidation of Isocitrate to α-Ketoglutarate and CO₂ In the next step, isocitrate dehydrogenase catalyzes oxidative decarboxylation of isocitrate to form α –ketoglutarate.

In this reaction isocitrate gives off a pair of hydrogen atoms (oxidation) and a molecule of CO₂ (decarboxylation) and becomes a 5-carbon α –ketoglutarate. The pair of Hydrogen atoms give two electrons and one H⁺ to NAD ⁺, forming NADH + H ⁺.

kreb’s cycle

• The Citric Acid Cycle Has Eight Steps:

4. Oxidation of α-Ketoglutarate to Succinyl-CoA and CO₂

• The next step is another oxidative decarboxylation, in which -ketoglutarate is converted to succinyl-CoA and CO₂ by the action of the -ketoglutarate dehydrogenase complex; NAD serves as electron acceptor and CoA as the carrier of the succinyl group.

kreb’s cycle

• The Citric Acid Cycle Has Eight Steps:

5. Conversion of Succinyl-CoA to Succinate

• In this step, Succinyl-CoA splits into 4- carbon succinate and Coenzyme A with the addition of water. The Coenzyme A tranfers its high energy (located at sulphur bond) to a phosphate group that joins GDP and forms GTP. The enzyme that catalyzes this reversible reaction is called succinyl-CoA synthetase

kreb’s cycle

• The Citric Acid Cycle Has Eight Steps:

6. Oxidation of Succinate to Fumarate

• The succinate formed from succinyl-CoA is oxidized to fumarate by the flavoprotein succinate dehydrogenase and liberates a pair of hydrogen atoms. The latter pass to FAD⁺, forming FADH₂. In eukaryotes, succinate dehydrogenase is tightly bound to the inner mitochondrial membrane; in prokaryotes, to the plasma membrane.

kreb’s cycle

• The Citric Acid Cycle Has Eight Steps:

7. Hydration of Fumarate to Malate

• The reversible hydration of fumarate to L-malate is catalyzed by fumarase in the presence of water.

kreb’s cycle

• The Citric Acid Cycle Has Eight Steps:

8. Oxidation of Malate to Oxaloacetate

• In the last reaction of the citric acid cycle, NAD-linked L-malate dehydrogenase catalyzes the oxidation of L-malate to oxaloacetate by removing a pair of hydrogen atoms from malate. The pair of hydrogen atoms pass two electrons and one H⁺ to NAD⁺, forming NADH + H⁺.
• Oxaloacetate combines with acetyl coenzyme A to form citrate and so the cycle continues.

Energetics of Kreb’s Cycle

Regulation of the Kreb’s Cycle/Citric Acid Cycle

• The citric acid cycle is regulated at various levels:
• 1. Production of Acetyl-CoA by the Pyruvate Dehydrogenase Complex.
• 2. Regulation at three exergonic steps i.e. entry of acetyl-CoA into the cycle (the citrate synthase reaction), Isocitrate dehydrogenase reaction and α-ketoglutarate dehydrogenase reaction.

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Regulation of the Kreb’s Cycle/Citric Acid Cycle

• 1. Regulation of Production of Acetyl-CoA by the Pyruvate Dehydrogenase Complex : This enzyme activity is turned off when ample fuel is available in the form of fatty acids and acetyl-CoA and when the cell has more ATP and NADH. On the other hand it is turned on when energy demands are high (i.e. when less amount of ATP and NADH are present) and the cell requires greater flux of acetyl-CoA into the citric acid cycle.
• Therefore the PDH complex is allosterically inhibited by ATP and by acetyl-CoA, NADH and long- chain fatty acids. While as AMP, CoA, and NAD allosterically activate the PDH complex.

Regulation of metabolite flow from the PDH complex through the citric acid cycle.

Regulation of the Kreb’s Cycle/Citric Acid Cycle

• PDH complex is also regulated by Covalent protein modification by reversible phosphorylation of E1 protein (pyruvate dehydrogenase) of PDH complex. When ample amount of ATP is available, a specific protein kinase phosphorylates and thereby inactivates E1.
• On the other hand when ATP declines, a specific phosphoprotein phosphatase removes the phosphoryl group by hydrolysis and thereby activates E1.

Regulation of the Kreb’s Cycle/Citric Acid Cycle

2. Regulation at three exergonic steps i.e. entry of acetyl-CoA into the cycle (the citrate synthase reaction), Isocitrate dehydrogenase reaction and α-ketoglutarate dehydrogenase reaction: These steps are regulated by substrate inhibition and allosteric feedback inhibition.

• i) Substrate availability inhibit the three reactions. For example, less availability of acetyl-CoA and oxaloacetate inhibit citrate synthase reaction. When NADH is present in high levels, malate dehydrogenase reaction slows down and results in slow production of oxaloacetate and thereby inhibits citrate synthase reaction. Also, citrate synthase and α-ketoglutarate dehydrogenase are inhibited by accumulation of NADH.
• ii) Product accumulation inhibits all three limiting steps of the cycle: succinyl-CoA inhibits - ketoglutarate dehydrogenase (and also citrate synthase); citrate blocks citrate synthase; and the end product, ATP, inhibits both citrate synthase and isocitrate dehydrogenase.

Regulation of the Kreb’s Cycle/Citric Acid Cycle

• iii) ADP acts as an allosteric activator of citrate synthase and isocitrate dehydrogenase.
• iv) Further, in vertebrate muscle, Ca²⁺, the signal for contraction and for a increase in demand for ATP, activates both isocitrate dehydrogenase and α–ketoglutarate dehydrogenase, as well as the PDH complex.

REFERENCES:

• Lehninger, A.L., Nelson, D.L. and Cox, M.M., 2005. Lehninger principles of biochemistry. Macmillan.
• Jain, J.L., 2004. Fundamentals of biochemistry. S. Chand Publishing.
• Satyanarayana, U., 2013. Biochemistry. Elsevier Health Sciences.