Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Citric Acid Cycle (CAC) AKA: Tricarboxylic Acid (TCA) Cycle and Krebs Cycle After this Lecture you will be able to answer: • For each step of the TCA cycle: – What do we get out of this? – Thermodynamics? – Is it Regulated? How? • Where do the carbons of Glucose become fully oxidized? • Why is the TCA cycle amphibolic? • How are TCA cycle intermediates replenished? Summary of Citric Acid Cycle Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi 2 CO2 + 3 NADH + 3H+ + FADH2 + GTP + CoA-SH Citric Acid Cycle Figure 17-2 Citrate Synthase (citrate condensing enzyme) CoA–SH O H3C C + S CoA Acetyl-SCoA C COOH H2C COOH O Oxaloacetate ∆Go’ = –31.5 kJ/mol H2C HO COOH C COOH H2C COOH Citrate Mechanism of Citrate Synthase (Formation of Acetyl-SCoA Enolate) Figure 17-10 part 1 Mechanism of Citrate Synthase (Acetyl-CoA Attack on Oxaloacetate) Figure 17-10 part 2 Mechanism of Citrate Synthase (Hydrolysis of Citryl-SCoA) Figure 17-10 part 2 Regulation of Citrate Synthase • Pacemaker Enzyme (rate-limiting step) • Rate depends on • Availability of substrates – Acetyl-SCoA – Oxaloacetate • Reduction Potential (NAD+/NADH) • Energy Charge (ATP/ADP/AMP)? • Inhibited by: – Citrate – Succinyl-CoA Aconitase ∆Go’ = ~0 H2C HO COOH H2O H2C COOH C COOH C COOH H2C COOH HC COOH Citrate (~91%) H2O H2C COOH HC COOH C H COOH HO Cis-aconitate (~3%) Isocitrate (~6%) Stereospecific Addition Iron-Sulfur Complex (4Fe-4S] Thought to coordinate citrate –OH to facilitate elimination Stereospecificity of Aconitase Reaction Prochiral Substrate Stereospecificity in Substrate Binding Page 325 Chiral Product NAD+–Dependent Isocitrate Dehydrogenase ∆Go’ = -20.9 kJ/mol NAD+ H2C COOH HC COOH C H COOH HO Isocitrate NADH + H+ H2C Mn2+ or Mg2+ COOH CH2 O C COOH -ketoglutarate Oxidative Decarboxylation NOTE: CO2 from oxaloacetate + CO2 Mechanism of Isocitrate Dehydrogenase (Oxidation of Isocitrate) Figure 17-11 part 1 Mechanism of Isocitrate Dehydrogenase (Decarboxylation of Oxalosuccinate) Mn2+ polarizes C=O Figure 17-11 part 2 Mechanism of Isocitrate Dehydrogenase (Formation of -Ketoglutarate) Figure 17-11 part 2 Regulation of Isocitrate Dehydrogenase • Pulls aconitase reaction • Regulation (allosteric enzyme) – Energy Charge (ATP/ADP) – Ca2+ • Competitive Inhibition – Reduction Potential (NADH/NAD+) • Accumulation of Citrate: – inhibits Phosphofructokinase Accumulation of Citrate CO2 Isocitrate dehydrogenase CO2 Isocitrate dehydrogenase -Ketoglutarate Dehydrogenase ∆Go’ = -33.5 kJ/mol NAD+ H2C COOH CH2 O C NADH + H+ H2C + CoASH COOH COOH CH2 O C SCoA Succinyl-CoA -ketoglutarate Oxidative Decarboxylation Mechanism similar to PDH CO2 from oxaloacetate High energy thioester + CO2 -Ketoglutarate Dehydrogenase (Multienzyme Complex) • E1: -Ketoglutarate Dehydrogenase or -Ketoglutarate Decarboxylase • E2: Dihydrolipoyl Transsuccinylase • E3: Dihydrolipoyl Dehydrogenase (same as E3 in PDH) Regulation of -Ketoglutarate Dehydrogenase • Competitive Inhibitors – NADH – Succinyl-SCoA • Activator: Ca2+ Origin of C-atoms in CO2 COOH H2C COOH C COOH HC COOH H2C COOH C H COOH H2C HO Citrate HO Isocitrate H2C COOH H2C CH2 O C COOH -ketoglutarate COOH CH2 O C SCoA Succinyl-CoA Both CO2 carbon atoms derived from oxaloacetate Succinyl-CoA Synthetase ∆Go’ = ~0 GDP + Pi H2C COOH CH2 O C SCoA Succinyl-CoA GTP H2C COOH H2C COOH + CoASH Succinate High Energy Thioester —> Phosphoanhydride Bond Plants and Bacteria: ADP + Pi —> ATP Randomizationn of labeled C atoms Thermodynamics (Succinyl-SCoA Synthetase) Succinyl-SCoA+ H2O GDP + Pi Succinyl-SCoA + GDP + Pi Succinate + CoA ² Go' = –32.6 kJ/mol GTP + H2O Go' = +30.5 kJ/mol ² Succinate + GTP + CoA ² Go' = –2.1 kJ/mol Mechanism of Succinyl-CoA Synthetase (Formation of High Energy Succinyl-P) Figure 17-12 part 1 Mechanism of Succinyl-CoA Synthetase (Formation of Phosphoryl-Histidine) Figure 17-12 part 2 Mechanism of Succinyl-CoA Synthetase (Phosphoryl Group Transfer) Substrate-level phosphorylation Figure 17-12 part 3 Nucleoside Diphosphate Kinase (Phosphoryl Group Transfer) GTP + ADP ——> GDP + ATP ∆Go’ = ~0 Which of the following is/are advantages of multienzyme complexes? A. They speed up reaction rates. B. Side reactions are limited by channeling C. The entire complex can be controlled as one unit. D. A and C E. All of the above Predict which of the following enzymes are identical in both the pyruvate dehydrogenase and the ketoglutarate dehydrogenase complexes. A. E1 B. E2 C. E3 D. E2 and E3 E. E1, E2 and E3 Which of the following enzymes catalyzes a reaction with the pictured compound as an intermediate? A. B. C. D. E. a-ketoglutarate dehydrogenase succinyl-CoA synthetase succinate dehydrogenase fumarase malate dehydrogenase Succinate Dehydrogenase ∆Go’ = ~0 FAD H2C COOH H2C COOH Succinate FADH2 CH HOOC HC COOH Fumarate Membrane-Bound Enzyme Direct entry into the ETC Covalent Attachment of FAD Figure 17-13 FAD used for Alkane Alkene • Reduction Potential (E) – Affinity for electrons; Higher E, Higher Affinity – Electrons transferred from lower to higher E FAD/FADH2 Reduction Potential Succinate/Fumarate NAD+/NADH Isocitrate/α-Ketoglutarate Fumarase ∆Go’ = ~0 H2O HOOC CH HC H C COOH H2C COOH HO COOH Fumarate Malate Malate Dehydrogenase NAD+ H C COOH H2C COOH HO NADH + H+ C COOH H2C COOH O Oxaloacetate Malate ∆Go’ = +29.7 kJ/mol Low [Oxaloacetate] Availability of NAD+ Thermodynamics Malate + NAD+ Oxaloacetate + NADH + H+ Acetyl-SCoA + Oxaloacetate Citrate + CoA Malate + NAD+ NADH + H+ + + Acetyl-SCoA Citrate + CoA ² Go' = +29.7 kJ/mol ² Go' = –31.5 kJ/mol ² Go' = –1.8 kJ/mol Products of the Citric Acid Cycle Figure 17-14 ATP Production from Products of the Central metabolic Pathway = 32 ATP NADH 2.5 ATP FADH2 1.5 ATP Page 584 1 6 Carbons of Glucose: 1st cycle 2 5 3 4 3, 4 2,5 1,6 1,6 2,5 2,5 1,6 1,6 2,5 2,5 1,6 Carbon Tracing from Glucose Carbon Tracing from Glucose • Glucose radiolabeled at specific Carbons – Can determine fate of individual carbons • Carbons 1,6 (Pyruvate Methyl) – 1st cycle: 2,3 of oxaloacetate – Starting at 3rd cycle ½ radioactivity CO2/cycle • Carbons 2,5 (Pyruvate Carbonyl) – 1st cycle: 1,4 of oxaloacetate – 2nd cycle: all eliminated as CO2 • Carbons 3,4 (Pyruvate Carboxyl) – All eliminated at CO2 during Pyruvate Acetyl-CoA You are following the metabolism of pyruvate in which the methyl-carbon is radioactive: *CH3COCOOH. -assuming all the pyruvate enters the TCA cycle as Acetyl-CoA, indicate the labeling pattern and its distribution in oxaloacetate first formed by operation of the TCA cycle. Amphibolic Nature of Citric Acid Cycle Generation of Citric Acid Cycle Intermediates Pyruvate Carboxylase ATP COOH ADP + Pi O H3C C CH2 COOH Pyruvate + HCO3– (CO2) O Pyruvate Carboxylase C COOH Oxaloacetate Animals and Some Bacteria In Mitochondrial Matrix Biotin Cofactor (CO2 Carrier) O HN H2 C C S NH O CH (CH2)4 C C O NH (CH2)4 CH NH Biotin Lysine Reaction Mechanism I (Dehydration/Activation of HCO3–) O AMP O O O P O P O– O– – O C OH O– ATP HN O HCO3 H2C – C S NH CH Biotinyl-Enzyme O (CH2)4 C NH (CH2)4 Enzyme ADP + Pi O O – O C N H2 C C S NH CH Carboxybiotinyl-Enzyme O (CH2)4 C NH (CH 2) 4 Enzmye Reaction Mechanism II (Transfer of CO2 to Pyruvate) O – O O C C CH 2 O – O – O Pyr uvate Enolate O – O O C C – C C N H 2C CH 2 S NH Car boxybiotinyl-Enzyme O CH (CH2) 4 C Biotinyl-Enzyme O – O O C C CH 2 C O O – Oxaloacetate NH (CH 2)4 Enzyme Fates of Oxaloacetate Gluconeogenesis ATP ADP + Pi COO– COO– C O + CH3 Pyruvate HCO3– C O Pyruvate Carboxylase CH2 COO– Oxaloacetate Regulation! Citric Acid Cycle Regulation of Pyruvate Carboxylase Allosteric Activator Acetyl-SCoA Glyoxylate Cycle Plants and Some Microorganisms In Glyoxysome Net Reaction of Glyoxylate Cycle 2 Acetyl-CoA 1 Oxaloacetate Net increase of one 4-carbon unit! Net conversion of acetyl-CoA to glucose Glyoxylate Cycle Enzymes H2 C COOH H2 C COOH HC COOH HO Isocitrate Lyase CH COOH Isocitrate H2 C COOH Succinate CHO + COOH Glyoxylate CoA-SH CHO COOH Glyoxylate + HO O H3C C S–CoA Acetyl–SCoA Malate Synthase CH COOH H2C COOH Malate Plants and Some Microorganisms Glyoxylate Cycle and the Glyoxysome Regulation of the Citric Acid Cycle Regulatory Mechanisms • Availability of substrates – Acetyl-CoA – Oxaloacetate • Product Inhibition – NADH • Energy Charge • Need for citric acid cycle intermediates as biosynthetic precursors • Demand for ATP Pyruvate Dehydrogenase Reaction and Regulation NAD+ NADH + H+ O CH3 C O COOH + CoenzymeA SH Pyruvate CH3 PDH (active) NADH Acetyl-CoA PDH Kinase Pyruvate ADP Ca2+ C S CoA + CO2 Acetyl–SCoA Insulin Ca2+ PDH Phosphatase PDH- P (inactive) Free Energy Changes of Citric Acid Cycle Enzymes Table 17-2 Regulation of Citrate Synthase • Pacemaker Enzyme (rate-limiting step) • Rate depends on • Availability of substrates – Acetyl-SCoA – Oxaloacetate • Reduction Potential (NAD+/NADH) • Energy Charge (ATP/ADP/AMP)? • Inhibited by: – Citrate – Succinyl-CoA Regulation of Isocitrate Dehydrogenase • Allosteric Enzyme – Energy Charge • ATP - inhibitor • ADP - activator – Reduction Potential • NADH - inhibitor • NAD+ - activator – Ca2+ Regulation of -Ketoglutarate Dehydrogenase • Inhibitors – NADH – Succinyl-SCoA • Activator: Ca2+ Regulation of the Citric Acid Cycle Figure 17-16 Shortened MCAT passage: The citric acid cycle starts with a two carbon acetyl-group from acetyl-CoA attaching to a four-carbon oxaloacetate molecule to produce a six-carbon citrate. During oxidative decarboxylation reactions, two carbons are lost as CO2. The citric acid cycle will result in a net gain of how many carbons? A. 0 carbons B. 1 carbon C. 2 carbons D. 4 carbons Which are the energy capture steps in the CAC? 8 A. B. C. D. E. 1, 3 and 4 1 and 5 3, 4, 6 and 8 5, 6 and 8 3, 4, 5, 6 and 8 1 7 2 6 3 5 4 If acetyl-CoA labeled with 14C, as shown in the figure to the right, were used as the substrate for the citric acid cycle, which of the following intermediates would be produced during the first round of the cycle? OH H O2*C C C CH2 CO2 H CO2 A) OH H O2C C C *CH2 CO2 H CO2 B) OH H O2C C) C C CH2 *CO2 H CO2 OH H O2C *C C CH2 CO2 D) H CO2 OH H O2C C *C CH2 CO2 E) H CO2 Germinating plant seeds can convert acetyl-CoA (obtained from fatty acids stored as oils) into carbohydrates, whereas animals are incapable of converting fatty acids into glucose. This difference is due to the fact that: A. animals have glycogen and don’t need to make glucose from fatty acids. B. plants use the glyoxylate cycle to convert two acetyl CoA to oxaloacetate, a precursor for gluconeogenesis. C. plant seeds use photosynthesis to make sugar. D. animals use the citric acid cycle selectively for energy production, whereas plants primarily use glycolysis. E. B and D • For each step of the TCA cycle: – What do we get out of this? – Thermodynamics? – Is it Regulated? How? • Where do the carbons of acetyl-CoA end up in the TCA cycle? • Why is the TCA cycle amphibolic? • How are TCA cycle intermediates replenished?