Lec-9-Metabolism-Fall 23.pptx.pdf

BTE 202: Metabolism Lecture-9 Citric Acid Cycle  Cellular respiration occurs in three major stages In the first stage, oxidation of organic fuel molecules- glucose, fatty acids and some amino acids occurs to produce acetyl-coenzyme A (acetyl-CoA). I...

BTE 202: Metabolism Lecture-9 Citric Acid Cycle  Cellular respiration occurs in three major stages In the first stage, oxidation of organic fuel molecules- glucose, fatty acids and some amino acids occurs to produce acetyl-coenzyme A (acetyl-CoA). In the next stage, the acetyl groups are fed into the citric acid cycle and enzymatically oxidized to CO2. The energy released during oxidation is conserved in the reduced electron carriers NADH and FADH2. The last stage is the electron transport chain where ATP is generated from the reduced electron carriers. Citric Acid or TCA or Kreb’s Cycle Citric Acid Cycle Step 1  Citric Acid Cycle Step 1: Formation of Citrate The first reaction of the cycle is the condensation of acetyl-CoA with oxaloacetate to form citrate, catalyzed by citrate synthase. FA oxidation Pyruvate Glucose Citric Acid Cycle Step 2  Citric Acid Cycle Step 2: Formation of Isocitrate via cis-Aconitate The enzyme aconitase (aconitate hydratase) catalyzes the reversible transformation of citrate to isocitrate, through the intermediary formation of the tricarboxylic acid cis-aconitate. Aconitase can promote the reversible addition of H2O to the double bond of enzyme-bound cis-aconitate in two different ways, one leading to citrate and the other to isocitrate. Citric Acid Cycle Step 3  Citric Acid Cycle Step 3: Oxidation of Isocitrate to α-Ketoglutarate and CO2 In the next step, isocitrate dehydrogenase catalyzes oxidative decarboxylation of isocitrate to form α-ketoglutarate. Citric Acid Cycle Step 4  Citric Acid Cycle Step 4: Oxidation of α-Ketoglutarate to Succinyl-CoA and CO2 The next step is another oxidative decarboxylation, in which α-ketoglutarate is converted to succinyl-CoA and CO2 by the action of the α-ketoglutarate dehydrogenase complex. NAD+ serves as electron acceptor and CoA as the carrier of the succinyl group. This reaction is similar to the pyruvate dehydrogenase complex reaction discussed eariler. α-ketoglutarate dehydrogenase complex closely resembles the PDH complex in both structure and function. Citric Acid Cycle Step 5  Citric Acid Cycle Step 5: Conversion of Succinyl-CoA to Succinate Succinyl-CoA, like acetyl-CoA, has a thioester bond with a strongly negative standard free energy of hydrolysis (∆G ≈ -36 kJ/mol). Energy released in the breakage of this bond is used to drive the synthesis of a phosphoanhydride bond in GTP. Citric Acid Cycle Step 6  Citric Acid Cycle Step 6: Oxidation of Succinate to Fumarate Succinate formed from succinyl-CoA is oxidized to fumarate by the succinate dehydrogenase. Malonate (an analog of succinate) is a strong competitive inhibitor of succinate dehydrogenase and its addition blocks the activity of the citric acid cycle. Citric Acid Cycle Step 7  Citric Acid Cycle Step 7: Hydration of Fumarate to Malate The reversible hydration of fumarate to L-malate is catalyzed by fumarase. Citric Acid Cycle Step 8  Citric Acid Cycle Step 8: Oxidation of Malate to Oxaloacetate In the last reaction of the citric acid cycle, L-malate dehydrogenase catalyzes the oxidation of L-malate to oxaloacetate.  Citric Acid or TCA or Kreb’s Cycle: A Set of 8 Reactions 1. Acetyl-CoA donates its acetyl group to the 4-C compound oxaloacetate to form the 6-C citrate with the help of Citrate Synthase. 2. Citrate is then transformed into isocitrate by Aconitase. 3. Isocitrate is dehydrogenated with loss of CO2 to yield the 5-C compound α-ketoglutarate by Isocitrate Dehydrogenase. 4. α-Ketoglutarate undergoes loss of a second molecule of CO2 and yields the 4-C compound succinyl-CoA by the action of α-Ketoglutarate Dehydrogenase Complex which functions and is regulated just in the same way as PDH complex. The oxidation of the carbons from acetyl-CoA is completed.  Citric Acid or TCA or Kreb’s Cycle: A Set of 8 Reactions These last 4 reactions regenerate the oxaloacetic acid 5. In the next step, Succinyl-CoA produces Succinate with the concomitant production of ATP from ADP and Pi by Succinyl-CoA Synthetase. [Substrate-level phosphorylation] 6. Succinate is then enzymatically converted into Fumarate by Succinate Dehydrogenase. Fumarate is the trans-isomer of Maleate. 7. Fumarate is specifically (not Maleate) hydrated (addition of H2O) into Malate in the penultimate step of TCA cycle by Fumarase. 8. And finally, Malate Dehydrogenase oxidizes Malate into Oxaloacetate which repeats the second cycle of these 8 reactions. tric Acid or TCA or Kreb’s Cycle: A Set of 8 Reactions At each turn of the cycle, one acetyl group (two carbons) enters as acetyl-CoA and two molecules of CO2 leave. One molecule of oxaloacetate is used to form citrate and one molecule of oxaloacetate is regenerated. Four of the eight steps in this process are oxidations, in which the energy of oxidation is very efficiently conserved in the form of the reduced coenzymes NADH and FADH2.  Energy of oxidations in the citric acid cycle In oxidative phosphorylation, passage of two electrons from NADH to O2 drives the formation of about 2.5 ATP, and passage of two electrons from FADH2 to O2 yields about 1.5 ATP. In round numbers, this represents the conservation of 32x30.5 kJ/mol 976 kJ/mol, or 34% of the theoretical maximum of about 2,840 kJ/mol available from the complete oxidation of glucose.  Here’s a list of all the ATP, NADH and FADH2 that are produced at different levels of the Carbohydrate Metabolic Pathway- Stage ATP NADH FADH2 Glycolysis 2 2 -- Acetyl-CoA Production -- 2 -- (From 2 molecules of Pyruvate) TCA Cycle 2 6 2 (From 2 molecules of Acetyl-CoA) 10 2 Electron Transport Chain (ETC) 10 x 2.5* = 25 ATP -- -- (From 10 NADH) 2 x 1.5* = 3 ATP (From 2 FADH2) Total 32 ATP * In the ETC, passage of two electrons from NADH to O2 drives the formation of about 2.5 ATP, and passage of two electrons from FADH2 to O2 yields about 1.5 ATP. Regulation of Citric Acid Cycle The citric acid cycle is regulated at its three exergonic steps These steps are catalyzed by- – Citrate synthase, – Isocitrate dehydrogenase, – α-ketoglutarate dehydrogenase ΔG´ᴼ = -8.4 kJ/mol The citric acid cycle is regulated at its three exergonic steps Availability of the substrates for citrate synthase (acetyl-CoA and oxaloacetate) varies with the metabolic state of the cell and limit the rate of citrate formation. Availability of the substrates  The citric acid cycle is regulated at its three exergonic steps If NADH (a product of isocitrate and α-ketoglutarate oxidation) accumulates (at high [NADH]/[NAD+]), both reactions are severely inhibited by mass action. FEEDBACK Inhibition The citric acid cycle is regulated at its three exergonic steps Citrate inhibits citrate synthase. Succinyl-CoA inhibits citrate synthase and α-ketoglutarate dehydrogenase. FEEDBACK Inhibition The citric acid cycle is regulated at its three exergonic steps ATP inhibits both citrate synthase and isocitrate dehydrogenase. The inhibition of citrate synthase by ATP is relieved by ADP (an allosteric activator of this enzyme). FEEDBACK Inhibition/ Allosteric Inhibition/ Energy Charge of Cell  The citric acid cycle is regulated at its three exergonic steps In vertebrate muscle, Ca2+(the signal for contraction and a concomitant increase in demand for ATP), activates both isocitrate dehydrogenase and α–ketoglutarate dehydrogenase, as well as the PDH complex. Allosteric Regulation/ Energy Demand of Cell  Regulation of citric acid cycle Su bs tra te s High [ATP] High [Acetyl-CoA] cts High [Succinyl-CoA] Citrate synthase High [Oxaloacetate] du Pro High [Citrate] High [ADP] High [NADH] Isocitrate High [α-ketoglutarate] dehydrogenase High [Ca2+] High [ATP] High [ATP] α-ketoglutarate High [NADH] dehydrogenase High [Ca2+] High [Succinyl-CoA] Concentrations of substrates and products in the citric acid cycle set the flux through this pathway at a rate that provides optimal concentrations of ATP and NADH. Summary of Regulation of TCA Cycle  Regulation of TCA Cycle The Citric Acid Cycle Is Regulated at Its Three Exergonic Steps!! First of all, α-Ketoglutarate Dehydrogenase Complex is regulated just in the same as is PDH Complex. Besides all these three irreversible reactions of the TCA cycle are regulated by FEEDBACK INHIBITION.  Regulation of TCA Cycle FEEDBACK INHIBITION: Which means- A high concentration of Citrate inhibits Citrate Synthase A high concentration of α-Ketoglutarate inhibits Isocitrate Dehydrogenase A high concentration of Succinyl-CoA inhibits α-Ketoglutarate Dehydrogenase Complex.  Regulation of TCA Cycle And finally, high concentration of the END PRODUCTS of this TCA cycle – ATP and NADH inhibits both Isocitrate Dehydrogenase and α-Ketoglutarate Dehydrogenase Complex SHUTTING DOWN the overall TCA cycle!!  Importance of TCA Cycle The citric acid cycle is central to the energy-yielding metabolism. But its role is not limited to energy conservation. Four- and five-carbon intermediates of the cycle serve as precursors for a wide variety of products.  Importance of TCA Cycle: Aerobic Organisms In aerobic organisms, the citric acid cycle is an amphibolic pathway, one that serves in both catabolic and anabolic processes. Besides its role in the oxidative catabolism of carbohydrates, fatty acids, and amino acids, the cycle provides precursors for many biosynthetic pathways, through reactions that served the same purpose in anaerobic ancestors. Importance of TCA Cycle: Aerobic Organisms  Importance of TCA Cycle: Anaerobic Organisms Early anaerobes probably used some of the reactions of the citric acid cycle in linear biosynthetic processes. Some modern anaerobic microorganisms still use an incomplete citric acid cycle as a source of biosynthetic precursors, but not as a source of energy. These organisms use the first three reactions of the cycle to make α-keto-glutarate but, lacking α-ketoglutarate dehydrogenase, (Therefore, cannot carry out the complete set of citric acid cycle reactions). They also have the four enzymes that catalyze the reversible conversion of oxaloacetate to succinyl-CoA and can produce malate, fumarate, succinate, and succinyl-CoA from oxaloacetate in a reversal of citric acid cycle. Importance of TCA Cycle: Anaerobic Organisms  Anaplerotic reactions replenish Citric Acid Cycle intermediates Anaplerotic reactions are those that form intermediates of a metabolic pathway. In aerobic organisms, the citric acid cycle is an amphibolic pathway- one that serves in both catabolic and anabolic processes. Intermediates of the citric acid cycle are used as biosynthetic precursors. These are replenished by anaplerotic reactions.  Anaplerotic reactions replenish Citric Acid Cycle intermediates Anaplerotic reactions are those that form intermediates of a metabolic pathway. The most important anaplerotic reaction is the reversible carboxylation of pyruvate by CO2 to form oxaloacetate, catalyzed by pyruvate carboxylase. When the citric acid cycle requires oxaloacetate, pyruvate is carboxylated to produce more oxaloacetate. The enzymatic addition of a carboxyl group to pyruvate requires energy, which is supplied by ATP. Anaplerotic reactions replenish Citric Acid Cycle intermediates Phosphoenolpyruvate (PEP) carboxylase Phosphoenolpyruvate (PEP) carboxylase is activated by the glycolytic intermediate fructose 1,6-bisphosphate, which accumulates when the citric acid cycle operates too slowly to process the pyruvate generated by glycolysis.  Anaplerotic Reactions Anaplerotic reactions are those that form intermediates of a metabolic pathway. Under normal circumstances, the reactions by which cycle intermediates are siphoned off into other pathways and those by which they are replenished are in dynamic balance, so that the concentrations of the citric acid cycle intermediates remain almost constant.  Enzymes of citric acid cycle may form multi-enzyme complexes Evidence suggests that within the mitochondrion the enzymes of the citric acid cycle exist as multi-enzyme complexes. Multi-enzyme complexes Separate enzymes  Substrate channeling through multienzyme complexes may occur in the citric acid cycle Substrate channeling is the passing of the intermediary metabolic product of one enzyme directly to another enzyme or active site without its release into solution. Multi-enzyme complexes ensure efficient passage of the product of one enzyme reaction to the next enzyme in the pathway. When several consecutive enzymes (enzyme complex) of a metabolic pathway channel substrates between themselves, this is called a metabolon. F E5 F E4 F D D C A soluble components D E1 A C E3 B B C E2 B Metabolon