Krebs Cycle Study Guide 2025 PDF

‫جامعة الملك سلمان الدولية‬ ‫كلية الطب‬ Krebs' Cycle Tricarboxylic Acid Cycle (TCA) Citric Acid Cycle (CAC)  TRICARBOXYLIC ACID CYCLE (Citric Acid Cycle or Krebs' Cycle) ILOs: By the end of the course, the student should be able...

‫جامعة الملك سلمان الدولية‬ ‫كلية الطب‬ Krebs' Cycle Tricarboxylic Acid Cycle (TCA) Citric Acid Cycle (CAC)  TRICARBOXYLIC ACID CYCLE (Citric Acid Cycle or Krebs' Cycle) ILOs: By the end of the course, the student should be able to: 1. Define tricarboxylic acid cycle. 2. Illustrate the steps of tricarboxylic acid cycle. 3. Point out the importance of tricarboxylic acid cycle. 4. Calculate the ATP generated per each turn of the cycle. 5. Explain how the tricarboxylic acid cycle is regulated. 6. Enumerate the inhibitors of the tricarboxylic acid cycle and explain their mechanism of action. -1-  TRICARBOXYLIC ACID CYCLE (Citric Acid Cycle or Krebs' Cycle) Definition: -The citric acid cycle is known as the Krebs' cycle or the tricarboxylic acid cycle (TCA cycle). It is formed of a series of reactions that are responsible for the complete oxidation of the acetyl moiety of acetyl-CoA. -It is the final common pathway for the oxidation of carbohydrates, lipids and proteins because glucose, fatty acids and all amino acids are metabolized to acetyl-CoA or intermediates of the cycle. -During the reactions of the cycle, coenzymes (NAD+ and FAD) are reduced and subsequently reoxidized by the respiratory chain with the formation of ATP. Site The enzymes of the TCA cycle are found in the mitochondrial matrix except succinate dehydrogenase which is tightly bound to the inner mitochondrial membrane (forms complex II of the respiratory chain). The enzymes of the TCA cycle are in close proximity to the enzymes of the respiratory chain. Steps of TCA Cycle 1- Formation of citrate The TCA cycle begins with the condensation of acetyl moiety of acetyl-CoA (C2) with oxaloacetate (C4) to form citrate (C6). The reaction is catalyzed by citrate synthase. 2- Formation of isocitrate Citrate is converted to isocitrate by the enzyme aconitase; the reaction occurs in two steps. Citrate is dehydrated to cis-aconitate then hydrated to isocitrate (C6). Aconitase contains iron-sulfur proteins and requires reduced glutathione. 3- Formation of α-ketoglutarate Isocitrate (C6) undergoes dehydrogenation catalyzed by isocitrate dehydrogenase to form initially, oxalosuccinate (C6), which remains enzyme bound, and undergoes decarboxylation to α-ketoglutarate (C5). This requires Mg2+ or Mn2+ for the decarboxylation reaction. This reaction produces the first NADH and releases the first CO2 of the cycle. 4- Formation of succinyl-CoA α-ketoglutarate (C5) undergoes oxidative decarboxylation to succinyl-CoA. This reaction is catalyzed by the enzyme α-ketoglutarate dehydrogenase complex. This complex requires the following cofactors; thiamine pyrophosphate (TPP), lipoate, CoASH, FAD and NAD+. This reaction produces the second NADH and releases the second CO2. 5- Formation of succinate Succinyl-CoA is converted to succinate (C4) by the enzyme succinate thiokinase. Succinate thiokinase cleaves the high-energy thioester bond of succinyl-CoA, the energy of which is used to generate the high energy phosphate bond of ATP. This is the only reaction in the TCA cycle that generates ATP by substrate level phosphorylation. -2- 6- Formation of fumarate Succinate (C4) is dehydrogenated to fumarate (C4) by succinate dehydrogenase which is bound to the inner mitochondrial membrane. The enzyme contains FAD (forms FADH2) and two iron sulfur centers. 7- Formation of malate Fumarate is hydrated to malate (C4) by the enzyme fumarase. 8- Regeneration of oxaloacetate Malate is dehydrogenated to oxaloacetate (C4) by the enzyme malate dehydrogenase. This produces the third NADH of the cycle. This reaction regenerates oxaloacetate, which condenses with a new acetyl molecule of acetyl – CoA to repeat the cycle again. Thus, the overall reactions of one turn of the Krebs' cycle yield two molecules of CO2, three molecules of NADH , one molecule of FADH2 and one molecule of ATP. Summary Diagram for TCA Cycle Citrate synthase Malate Aconitase dehydrogenase Isocitrate Fumarase dehydrogenase Succinate -Ketoglutarate dehydrogenase dehydrogenase Succinate -3-  CH3– CO ~ S-CoA Active acetate CoA- SH CH2– COOH O=C- COOH HO–C– COOH CH2-COOH CH2– COOH Citrate synthase Oxaloacetate H2O Citrate Aconitase NADH,H+ Fe2+ Malate dehydrogenase G-SH H2O NAD+ TCA Cycle CH2– COOH HO–CH–COOH C– COOH CH2– COOH CH– COOH L-Malate Cis-aconitate Fumarase Aconitase H2O H2O Fe2+ G-SH HC – COOH CH2– COOH HOOC – CH ETC 10 ATP HC– COOH Fumarate HOCH– COOH Isocitrate FADH2 Succinate NAD+ dehydrogenase Isocitrate FAD dehydrogenase CH2– COOH NADH,H+ CH2– COOH CH2– COOH Succinate HC– COOH CoA-SH ATP O=C – COOH Succinate thiokinase Oxalosuccinate Mg2+ Isocitrate ADP + Pi dehydrogenase Mn2+ CO2 CH2– COOH -Ketoglutarate CoA-SH NADH,H+ NAD+ dehydrogenase CH2– COOH CH2 complex CH2 O=C ~ S-CoA TPP, Lipoate, & FAD O=C – COOH Succinyl-CoA CO2 -Ketoglutarate -4- Importance of Citric Acid Cycle The citric acid cycle is called an amphibolic pathway because it participates in both catabolism and anabolism. I- Energy production The oxidation of one mole of the acetyl group of acetyl-CoA through Krebsʹ cycle yields 10 moles of ATP. ETC 3 NADH, H+ 7.5 ATP ETC FADH2 1.5 ATP Substrate level ADP + Pi ATP Total 10 ATP II- It is a common final pathway for oxidation of carbohydrates, fats and amino acids -5- III- Importance of citric acid cycle intermediates (anabolic function) Many of the intermediates produced in the citric acid cycle are precursors for important biomolecules (anabolic function). 1- Citrate Citrate can be exported out of the mitochondria into the cytosol where it is broken down by ATP-citrate lyase to yield oxaloacetate and acetyl-CoA. The acetyl-CoA produced is a precursor for fatty acids and cholesterol. 2- α-ketoglutarate α-ketoglutarate is converted by amino transferase into glutamate, which is an important amino acid. 3- Succinyl-CoA It is used for heme synthesis and oxidation of ketone bodies (Ketolysis). 4- Malate Malate can be reoxidized into oxaloacetate or can be oxidatively decarboxylated to pyruvate by malic enzyme (it is one of the sources of NADPH). Malic Enzyme COOH HOCH-COOH C=O CH2-COOH Malate NADP+ NADPH,H+ CH3 + CO2 Pyruvate 5- Oxaloacetate Oxaloacetate can be transaminated by aspartate aminotransferase (AAT or AST) to form aspartate, which is an important amino acid. In cytosol, oxaloacetate is converted into 2-phosphoenolpyruvate (2-PEP) by phosphoenolpyruvate carboxykinase enzyme (PEPCK), which is an important step in gluconeogenesis (formation of glucose from non-carbohydrate compounds). IV. Importance of CO2 CO2 produced by isocitrate dehydrogenase and α-ketoglutarate dehydrogenase is used in many important reactions : 1- Conversion of pyruvate to oxaloacetate by pyruvate carboxylase. 2- Conversion of acetyl -CoA to malonyl-CoA, which is used in FA synthesis. 3- Conversion of propionyl-CoA to methylmalonyl-CoA. 4- Synthesis of carbamoyl-phosphate for synthesis of pyrimidines and urea. 5- Formation of C6 of purines. 6- Formation of H2CO3/ BHCO3 buffer system. V. Interconversion between carbohydrates, fats and proteins TCA cycle represents the center of body metabolism. Through the cycle intermediates, carbohydrates, fats and amino acids are interconverted during the feed-starve cycle. -6-  Importance of the Intermediates of TCA Cycle (Anabolic Function) Cholesterol Cytosolic ATP – Citrate Lyase Citrate Acetyl - CoA CoA Oxaloacetate ATP ADP + Pi Fatty acids Transamination -Ketoglutarate Glutamate Heme Succinyl-CoA Ketolysis Cytosolic malic enzyme Malate Pyruvate NADP+ NADPH,H+ + CO2 Cytosolic PEPCK Oxaloacetate 2-Phosphoenolpyruvate GTP GDP + CO2 Gluconeogenesis AST Aspartate Glucose -7- Regulation of Citric Acid Cycle The citric acid cycle must be carefully regulated by the cell. If the citric acid cycle was permitted to run unchecked, large amounts of metabolic energy would be wasted in the over production of reduced coenzymes and ATP. Conversely if the citric acid cycle ran too slowly, ATP would not be generated fast enough to sustain the cell. In the Krebs' cycle there are three irreversible steps. These three reactions of the cycle are catalyzed by citrate synthase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase which are the rate controlling enzymes of the cycle (key enzymes). The TCA is regulated through as follows: A. Substrate activation and product feedback inhibition 1-The availability of both oxaloacetate and acetyl-CoA is very important for the activity of citrate synthase. - Oxaloacetate is formed from pyruvate by pyruvate carboxylase enzyme. The latter is allosterically activated by acetyl-CoA, this ensures an enough supply of oxaloacetate for the activation of the cycle. - The mitochondria contain a very low concentration of oxaloacetate (lower than the Km value of oxaloacetate on citrate synthase). Therefore, any increase in the concentration of oxaloacetate increases the activity of citrate synthase to a great extent. 2- Increased concentration of succinyl-CoA produces feedback inhibition of citrate synthase and α-ketoglutarate dehydrogenase. Diagram for Regulation of TCA Cycle Active Acetate + Pyruvate Citrate synthase ADP & Ca2+ (muscles) carboxylase + Pyruvate Oxaloacetate + Citrate - - L-Malate ADP & NAD Ca2+ (muscles) Fumarate (C4) ATP Isocitrate + Succinate - Isocitrate dehydrogenase - - -Ketoglutarate - Succinyl~S-CoA ADP & NAD -Ketoglutarate - + NADH Ca2+ (muscles) dehydrogenase -8- B- NADH/NAD+ ratio High NADH/NAD+ ratio inhibits isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase and vice versa. This mechanism for regulation is due to inhibition by NADH of the enzymes that use NAD+ and produce NADH. This mechanism explains that the cycle is only active under aerobic condition, where ETC is active to oxidize NADH into NAD+. C- ATP/ ADP ratio (Energy Requirements) The rate of the Krebs' cycle reflects the cell's need for ATP. When ATP/ ADP ratio is high, the rate of the citric acid cycle is reduced. High ATP/ADP ratio inhibits citrate synthase, isocitrate dehydrogenase and alpha- ketoglutarate dehydrogenase and vice versa. So, TCA cycle is turned on by high ratios of ADP/ATP or NAD+/NADH and vice versa. During Work ↓ATP → ↑ ADP → Activation of ATP synthase → Activation of oxidation by ETC → ↓NADH → ↑NAD → Activation of TCA cycle key enzymes During Rest ↑ATP → ↓ADP → Inhibition of ATP synthase → Inhibition of oxidation by ETC → ↑NADH → ↓NAD → Inhibition of TCA cycle key enzymes D- Calcium (Effect of muscular exercise): Calcium release from sarcoplasmic reticulum is increased to trigger muscle contraction. Ca2+ is transported into mitochondria by specific Ca2+ transporters through the outer and inner mitochondrial membrane. Within the mitochondria, Ca2+ activates allosterically citrate synthase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase to supply the contracting muscles with ATP. Inhibitors of Citric Acid Cycle 1. Fluroacetate inhibits activity of aconitase. It is used as rodenticide. In the body, it is converted to fluoroacetyl-CoA, which gives rise to fluorocitrate after condensing with oxaloacetate. The aconitase is inhibited by fluorocitrate. 2. α-ketoglutarate dehydrogenase is inhibited by arsenic compounds. Arsenite forms a stable complex with the thiol groups of lipoic acid making it unavailable for the enzyme action. 3. Malonate is a competitive inhibitor of succinate dehydrogenase. -9-