Steps of Krebs cycle Krebs cycle is a stepwise cyclic oxidation process in which four dehydrogenation steps and two

decarboxylations steps of active acetate group takes place to produce reduced co-enzymes and Carbon Dioxide. The following nine steps occur in overall Krebs cycle: 1. 2. 3. 4. 5. 6. 7. 8. 9. Condensation Isomerisation Dehydrogenation Decarboxylation Oxidative Decarboxylation Substrate level ATP/GTP synthesis. Dehydrogenation (oxidation) of Succinate Hydration and Dehydrogenation (Oxidation) of Malate

Citric Acid or Krebs cycle completed in Nine steps Step 1: Condensation In first step of Krebs cycle, Acetyl CoA combines with oxaloacetate in the presence of condensingenzymes citrate synthetase. CoA is released out. The product of condensation is citrate which is a tricarboxylic 6carbon compound. Step 2: Isomerisation Citrate formed in first step is converted into its isomer isocitrate in a two step reaction in the presence of iron containing enzyme aconitase. (i) Dehydration : A molecule of water is released and citric acid is changed into cis-aconitate. (ii) Rehydration : Cis aconitate combines with a molecule of water and form isocitrate. Step 3: Dehydrogenation Now isocitrate undergoes dehydrogenation in the presence of an enzyme isocitrate dehydrogenase. Mn2+ ion is required for the functioning of enzyme. Hydrogen given out by isocitrate is picked up by NAD+ (Nicotinamide adenine dinucleotide) to form NADH2. After losing hydrogen, isocitrate is changed into oxalosuccinate (6C). Step 4: Decarboxylation Oxalosuccinate in Step 4 undergoes decarboxylation. In the presence of oxalosuccinate decarboxylase enzyme, oxalosuccinate is changed into -ketoglutarate. Step 5: Oxidative Decarboxylation In this step 5-carbon compound, Ketoglutarate undergoes simultaneous dehydrogenation and decarboxylation in the presence of enzyme ketoglutarate dehydrogenase complex. This enzyme complex contain TPP, Lipoic Acid , Mg2+ and trans succinylase. NAD+ and CoA are required. The products formed are 4 carbon compound succinyl CoA, NADH2 and CO2. Step 6: Substrate level ATP/GTP Synthesis In the presence of enzyme succinyl thiokinase, succinyl CoA is hydrolyzed. CoA and Succinate are formed. The energy liberated during the process is used in synthesis of ATP in Plants and GTP (Guanosine triphosphate) or ITP (Inosine triphosphate) in animals. CoA is released out. Step 7: Dehydrogenation (Oxidation) In step 7 of Krebs Cycle 4 Carbon compound Succinate is oxidized to another 4-carbon compound fumarate with the help of enzyme succinate dehydrogenase and hydrogen acceptor FAD (Flavin Adenine Dinucleotide). The enzyme is attached to inner mitochondrial membrane. It contains or non haem iron (FeS) protein. This enables the enzyme to get directly linked to electron transport chain. Step 8: Hydration

In step 8, Fumarate reacts with a molecule of water, in the presence of an enzyme fumarase forming another 4-carbon dicarboxylic acid called Malate. Step 9: Dehydrogenation (Oxidation) With the help of enzyme malate dehydrogenase, Malate formed in step 8 is oxidized to oxaloacetate. NAD+ reduced to NADH2. An oxaloacetate formed in this reaction becomes available to combine with acetyl CoA to start a new cycle all over again. Note : The overall equation of oxidative catabolism of pyruvate can be written as follows:NADH2 & FADH2 are linked to electron transport system and formation of ATP by Oxidative Phosphorylation. Sites for Carbon Dioxide Production Carbon Dioxide is not formed during glycolysis. Three molecules of Carbon Dioxide are evolved during complete oxidation of each of the two pyruvates. One molecule is produced during link reaction when Oxidative Decarboxylation of pyruvate to acetyl CoA takes place. Two molecules are produced during Krebs cycle: One during Decarboxylation of Oxalosuccinate to Ketoglutarate and another during Decarboxylation of Ketoglutarate to succinyl CoA. Sites for Substrate level Phosphorylation Four molecules of ATP are formed through substrate level phosphorylation in glycolysis. (i) Two during dephosphorylation of two molecules of 1, 3 diphosphoglyceric acid to two molecules of 3 phosphoglyceric acids. (ii) Two during dephosphorylation of two molecules of phosphoenol pyruvate to the two molecules of pyruvate. Two molecules of ATP/GTP/ITP are formed through substrate level phosphorylation which is linked to release of energy at the time of breaking thioester bonds of two molecules of succinyl CoA to succinate state. Sites for Reduced Co-enzymes (i) Two molecules of NADH (+H+) are formed in glycolysis during oxidation of two molecules of glyceraldehydes -3- phosphate to 1, 3 diphosphoglycerate state. (ii) Two Molecules of NADH (+H+) are produced in link reaction or gateway step when two pyruvate molecules are oxidatively decarboxylated to the state of acetyl CoA. (iii) In Krebs Cycle six molecules of NADH (+H+) and two molecules of FADH2 are formed, the break up of which is

Oxidation of isocitrate to oxalosuccinate 2 NADH (+H+) Oxidative decarboxylation of Ketoglutarate to form succinyl CoA 2NADH (+H+). Dehydrogenation of succinate to form fumarate FADH2 Dehydrogenation of malate to oxaloacetate 2 NADH (+H+).

The net number of coenzymes formed from one molecule of glucose are 10 NADH (+H+) and 2FADH2. There is also a gain of 4 ATP molecules. Krebs cycle: Important Facts 1. 2. 3. 4. 5. 6. 7. Citric Acid cycle is the second step of cellular respiration. Its substrate is acetyl CoA. Activated acetate of acetyl CoA is completely broken down to inorganic state. It generally occurs inside mitochondria (except aerobic prokaryotes). Krebs cycle is restricted to only aerobic respiration. It is a cyclic pathway. It requires a link reaction or gateway step.

8. 9.

Krebs cycle does not consume any ATP molecules. It generates 2ATP/GTP/ITP molecules through substrate level Phosphorylation.

10. Krebs cycles produces 6 NADH (+H+) and two FADH2 molecules. 2NADH (+H+) are additionally formed through link reaction. 11. It is linked to electron transport. 12. It produces CO2. 13. Enzymes of Krebs cycle are located both in the matrix and inner membrane of mitochondrion. Krebs Cycle: Significance 1. 2. By this cycle, carbon skeleton are got, which are used in process of growth and for maintaining the cells. Intermediate compounds formed during Krebs cycle are used for the synthesis of biomolecules like amino acids, nucleotides, chlorophyll, cytochromes and fats etc. Intermediate like succinyl CoA takes part in the formation of chlorophyll. Amino Acids are formed from - Ketoglutaric acid, pyruvic acids and oxaloacetic acid. Krebs cycle (citric Acid cycle) releases plenty of energy (ATP) required for various of cell. metabolic activities

3. 4. 5.