The Citric Acid Cycle Lecture 4

The Citric Acid Cycle: Lecture 4 The Citric Acid Cycle: Fumarase sp 2 stereo-specific • Stereospecific – Addition of water is always trans and forms L-malate. – OH− adds to fumarate… then H+ adds to the carbanion. – Cannot distinguish between inner carbons, so either can gain –OH • Slightly thermodynamically favorable/reversible (DG°’ = –0.9 kcal/mol). – product concentration kept low to pull reaction forward The Citric Acid Cycle: Fumarase Mechanism ene-olic intermediate Recall enolase & aconitase H C HO– –OOC COO– H+ C H The Citric Acid Cycle Citrate Synthase Aconitase ICDH & aKGDH Suc-CoA Synthetase SucDH Fumarase ✓ ✓ ✓ ✓ ✓ ✓ glutarate a-ketoglutarate à succinate à fumarate à malate à oxaloacetate | citrate à aconitate à isocitrate “pyruvate” (acetate) The Citric Acid Cycle: Malate Dehydrogenase (OMSGAP) Oxidation of Alcohol to a Ketone and Regeneration of Oxaloacetate • Final step of the cycle • Regenerates oxaloacetate (a-ketosuccinate) for citrate synthase • Highly thermodynamically UNfavorable (DG°’ = +7.1 kcal/mol). – Similar to LDH & ADH – Reversible – oxaloacetate concentration kept VERY low by citrate synthase • pulls the reaction forward (–7.7 + 7.1 = –0.6) The Citric Acid Cycle Citrate Synthase Aconitase ICDH & aKGDH Suc-CoA Synthetase SucDH Fumarase Malate DH ✓ ✓ ✓ ✓ ✓ ✓ ✓ glutarate a-ketoglutarate à succinate à fumarate à malate à oxaloacetate | citrate à aconitate à isocitrate “pyruvate” (acetate) The Citric Acid Cycle Origin of C-Atoms in CO2 H2C COOH H2C COOH C COOH HC COOH H2C COOH C H COOH HO Citrate HO DG°’ = -7.7 +3.2 -7.7 -4.5 H2C H2C COOH CH2 O Isocitrate C COOH O C SCoA -7.2 -13.7 -0.7 -14.4 C C H C C H 2C C H 2 C C H Malate Succinyl-CoA a-ketoglutarate -2.0 -6.5 COOH CH2 0 -14.4 -0.9 -15.3 Oxaloacetate +7.1 -8.2 each step cumulative Energetics: why the cycle only turns in one direction • Carbons from acetate are red. • All CO2 generated during the citric acid cycle is produced before succinylCoA is made. • In one turn of the citric acid cycle, neither of the acetyl-CoA carbons is lost. • Both of the CO2 molecules lost were present on the oxaloacetate used to begin the cycle. • Overall energetics makes cycle irreversible: –Citrate synthase, IDH, and KDH – what about kinetics? • Nature of a cycle; acts as a unit and goes as fast as the available carbon The Citric Acid Cycle Citrate Synthase Aconitase ICDH & aKGDH Suc-CoA Synthetase Succinate DH Fumarase Malate DH ✓ ✓ ✓ ✓ ✓ ✓ ✓ Pyruvate dehydrogenase Acetyl CoA Krebs Cycle The Citric Acid Cycle Regulation* aconitase *in mammals The Citric Acid Cycle Regulation* •General regulatory mechanism – activated by substrate availability – inhibited by product accumulation (acetyl-CoA, succinyl-CoA, citrate) – Overall products of the pathway are NADH and ATP. • affects all regulated enzymes in the cycle • inhibitors: NADH and ATP • activators: NAD+, ADP, and AMP •Citrate synthase is also inhibited by succinylCoA. Classic feedback inhibition aconitase – α-Ketoglutarate is an important branch point for amino acid metabolism. – Succinyl-CoA communicates flow at this branch point to the start of the cycle. •Regulation of isocitrate dehydrogenase controls citrate levels; important for fatty acid metabolism. Energy Charge. *in mammals – Aconitase is reversible. – Inhibition of IDH leads to accumulation of isocitrate and reverses aconitase. – Accumulated citrate leaves mitochondria and inhibits phosphofructokinase in glycolysis. The Citric Acid Cycle Yield (TCA): Acetyl-CoA + 3NAD+ + FAD + GDP + Pi + 2 H2O à 2CO2 + 3NADH + FADH2 + GTP + CoA + 3H+ Yield (from pyruvate): Pyruvate + 4NAD+ + FAD + GDP + Pi + 2 H2O à 3CO2 + 4NADH + FADH2 + GTP + 3H+ From acetyl-CoA: • Net oxidation of two carbons to CO2 NADH Pyruvate CO2 – equivalent to two carbons of acetylCoA – but NOT the exact same carbons • Energy captured by electron transfer to NADH and FADH2 • Generates 1 GTP, which can be converted to ATP • Cycle acts as a unit; its basically a furnace for burning carbon 3 6 3 3 3 3 ….....except we don’t have the water yet! The Citric Acid Cycle: Summary 4Fe4S TPP Lipoic acid FAD Mn2+ NAD+ FeS isocitrate Mn+2 FAD NAD+ This slide has everything you need to commit to memory NAD+ Pi TPP Lipoic acid FAD 8 steps 8 enzymes 9 structures 13 cofactors /co-substrates Pyruvate Oxidation & Citric Acid Cycle Summary We learned that: • a large multi-subunit enzyme, pyruvate dehydrogenase complex, converts pyruvate into acetyl-CoA • several cofactors are involved in reactions that harness the energy from pyruvate • the citric acid cycle is an important catabolic process: it makes reduced cofactors (NADH & FADH2), plus GTP, that could yield ATP • the rules of organic chemistry help to rationalize reactions in the citric acid cycle • the citric acid cycle is largely regulated by availability of substrates and product inhibition (especially NADH and ATP) Oxidative Phosphorylation Oxidative Phosphorylation Glycogenolysis Glycolysis Pyruvate Oxidation Krebs' Cycle Phosphorylation Oxidative Oxidative Phosphorylation Learning goals: • Function of electron-transport chain in mitochondria…... make water (finish the reaction: C6H12O6 + 6O2 à 6CO2 + 6H2O) • Building up the proton-motive force • Synthesis of ATP in mitochondria, chloroplasts, & bacteria • Fuels for the cell, in the form of reduced carbon compounds (sugars), have been burned to carbon dioxide. • Electrons from reduced fuels are transferred to cofactors, which get reduced as NADH or FADH2. • In oxidative phosphorylation, energy from NADH …... But how? and FADH2 is used to make ATP. Clinical Correlations Hemolytic Anemia: Deficiencies in Glycolytic Enzymes • Red-blood cells do not have nuclei or mitochondria. They still need to maintain their membrane potential using the Na/K pump and membrane shape using actin microfilaments. Both use glycolysis as the sole source of ATP. • Any glycolytic enzyme that is impaired sufficiently will affect the efficiency of • The precise defect in aldolase A that causes hemolytic anemia the entire pathway. Without ATP, the lead to a deeper understanding cells swell and lyse, which is a of protein stability and cellular condition called non-spherocytic function. hemolytic anemia. • The most common form is from a deficiency in pyruvate kinase. This form is more tolerable than a form from a deficiency of aldolase A. This is likely because there is a buildup of intermediates behind the block at PK, including 2,3,bisphosphoglycerate, the negative heterotropic allosteric effector of Hb. This causes Hb to release more O2. Clinical Correlations Lactic Acidosis • This term refers to the acidification of the blood. Blood pH dropping to anything below 7.40 causes hyperventilation, lethargy, disorientation, etc. • Lactate is the major cause of acidosis, although any acidic metabolic intermediate, if accumulating too high, will cause this acidosis. Lactate often goes from 1.2 mM to 5 mM in the blood. • The cause of blood [Lactate] increase can be varied, but normally it’s from inadequate oxygenation. Muscle tissue easily generates large quantities of lactate, but the Cori cycle normally keeps homeostasis by converting it back to glucose through gluconeogenesis, which takes mitochondria-generated ATP. Other tissues will use the lactate and convert it back to pyruvate for combustion to CO2 and water, which is also dependent on oxygen. • Finding and treating the underlying causes of the acidosis is the challenge for physicians. Otherwise, all they can do is treat the symptoms: Bicarbonate administration • For example, treatments for diabetes often involve inhibition of gluconeogenesis (Metformin) or OxPhos (phenformin). • Thiamin deficiency (alcoholics) Clinical Correlations Fructose Metabolism: Malabsorption, Essential Fructosuria & Hereditary Fructose Intolerance • Three maladies arise due to fructose metabolism. • Only the last one, deficiency of aldolase B, can lead to death. Even small amounts of fructose lead to severe hypoglycemia, jaundice, hemorrhage, hepatomegaly, uricemia, and kidney damage. • Fructose can account for 30-60% of carbohydrate in the Western diet. Humans have a limited capacity to handle this sugar, and show the same ATP depletion after a sugar bolus. Use of fructose, sorbitol, and xylitol for parenteral nutrition Fructose (i.v.) has been Fructose GLUT2/ discontinued to to liver Malabsorption GLUT5 1 in 3 damage. mitochondrial OxPhos Fructose ATP Ketohexokinase ADP Hepatic Fructokinase Deficiency (aka Essential Fructosuria) 1 in 130,000 Fructose-1-phosphate Aldolase B AMP Hereditary Fructose Intolerance 1 in 20,000 Glyceraldehyde DHAP IMP G3P Uric Acid Giltzelmann et al., 1995