Introduction to the Krebs Cycle Hans Kreb discovered its cyclic nature Goes by three names – Citric acid cycle – Tricarboxylic cycle – Krebs cycle

9.2 A Cyclic Pathway Linear pathways can also be viewed as cyclic – Example – ethanol from pyruvate in yeast Pyruvate decarboxylase has several forms Pyruvate enters cycle Products CO 2 and acetaldehyde leave Acetaldehyde and NAD then enter and NADH and ethanol leave – Every enzymatic reaction can be written as a cycle One molecule of an enzyme can catalyze infinitely many conversions

9.2 Acetyl CoA: Substrate of the Krebs Cycle Overall process: oxidation of two acetyl carbons of acetyl-CoA completely to CO 2 – high energy electrons are captured Acetyl CoA – Thioester of acetate with CoA – Free SH group Formation of acetyl – CoA from pyruvate requires enzyme complex Occurs in mitochondria Cytosolic pyruvate must cross two membrane of mitochondria – Outer membrane can be crossed using porins – Inner membrane can be crossed using transport proteins

9.2 Acetyl CoA: Substrate of the Krebs Cycle Pyruvate dehydrogenase complex – Enzyme complexes are groups of enzyme that catalyze a metabolic sequence without releasing intermediates – Involves 3 separate enzymes – Five cofactors Mobile cofactors – CoA, NAD and NADH Bound cofactors – thiamine pyrophosphate, lipoic acid and flavin adenine dinucleotide

9.2 Acetyl CoA: Substrate of the Krebs Cycle Overall reaction is pyruvate + CoA + NAD+ which produce Acetyl-CoA and NADH Mechanism – E1 first reacts with – E2 froms acetyl-thioester and regenerates E1 – Mobile cofactor CoA-SH displaces acetyl group from E2 – E2 needs to be regenerated – FAD is regenerated using mobile cofactor NAD

9.3 Overview of Carbon Flow Intermediates of Krebs cycle are six, five and four carbon compounds Input to pathway is 2 carbon acetyl CoA condenses with 4 carbon compound First CO 2 is lost in the subsequent reactions - CO2 is from 4 carbon skeleton, not the incoming acetyl CoA

9.3 Overview of Carbon Flow Second CO2 is lost in another reaction – Again from the carbon skeleton and not the incoming acetyl CoA Resulting 4 carbon intermediate is converted to a molecule with plane of symmetry

9.4 Steps of the Pathway Citrate Synthase reaction – Introduces new carbon into pathway – Overall reaction – oxaloacetate + acetyl CoA produces citrate and CoA – Metabolically irreversible – No allosteric modulators

9.4 Steps of the Pathway Citrate Synthase reaction cont. – Mechanism – Extraction of a proton from terminal methyl group of acetyl CoA Nucleophilic substitution leads to citryl-CoA intermediate Hydrolyzed to citrate

9.4 Steps of the Pathway Aconitase – Overall reaction – citrate is dehydrate to cis- aconitate and then rehydrated to isocitrate – Mechanism Requires Fe-S cluster » Fe 2+ ions play a dual role Hydroxyl group attacks carbon 5 » Produces isocitrate

9.4 Steps of the Pathway Aconitase cont – Creates chiral carbon Enzyme has a three point landing Prochiral center – Identifies a carbon with two identical substituents that can be discriminated

9.4 Steps of the Pathway Fluoroacetate Poisoning involves the first two enzymes of the Krebs Cycle Fluoroacetate – active ingredient in poisons used in rodenticide Enzymatic conversion to an inhibitor – citrate synthaste reactions creates fluorocitrate – Fluorocitrate is irreversible inhibitor of aconitase

9.4 Steps of the Pathway Isocitrate dehydrogenase – Overall reaction – oxidation of isocitrate to produce 2-ketoglutarate NAD is used as a mobile cofactor CO 2 is also released – Metabolically irreversible reaction

9.4 Steps of the Pathway Isocitrate dehydrogenase cont. – Mechanism Oxidation to chemically unstable beta keto acid intermediate Decarboxylation Enol- keto form are in equilibrium

9.4 Steps of the Pathway -Ketoglutarate DH Complex – Overall reaction – 2 ketoglutarate reacts with acetyl CoA and NAD to produce succinyl CoA, NADH and CO 2 – Enzyme complex – Similar mechanism to pyruvate dehydrogenase complex – Metabolically irreversible – Important regulatory site of Krebs cycle

9.4 Steps of the Pathway Succinyl – CoA synthetase – Can also be called succinate thiokinase – Overall Reaction - Succinyl CoA reacts with GDP and Pi to form succinate, GTP and CoA-SH – Only reaction in Krebs cycle to form high energy phosphate – Metabolically irreversible reaction

9.4 Steps of the Pathway Succinyl CoA synthetase cont. – Mechanism Displacement of thioester by inorganic phosphate High energy phosphate is transferred to histidyl group High energy phosphate is transferred from the histidyl group – GTP can be directly used as energy intermediate

9.4 Steps of the Pathway Succinate Dehydrogenase – Overall reaction – conversion of succinate to fumarate – Only membrane in Krebs cycle that is membrane bound – Ubiquinone is used as a redox cofactor – – Metabolically irreversible No known regulators

9.4 Steps of the Pathway Succinate dehydrogenase cont – Enzyme complex Catalyzes series of electron transfers Electrons are removed from the interior C-C bond Using ubiquinone as electron carrier – Also plays a role in oxidative phosphorylation – Inhibitor – dicarboxylate malonate –

9.4 Steps of the Pathway Fumarase – Overall reaction – hydration of fumarate to malate – Near equilibrium enzyme – Mechanism Adds water across the C-C double bond – New chiral center is created Has Fe-S complex for substrate positioning

9.4 Steps of the Pathway Malate dehydrogenase – Overall reaction – malate is converted to oxaloacetate NAD is used as a mobile cofactor – Near equilibrium reaction – Mechanism Transfer of hydride from C-H bond to NAD ring – Oxaloacetate product is achiral Reverse reaction creates a chiral malate

9.5 Energy Balance Assumptions can be made as to how much energy is extracted by energy cofactors – NADH = 3 ATP – FADH 2 = 2 ATP – GTP = 1 ATP Using these assumptions, we can estimate a total of 12 ATP per cycle – 3 NAD linked dehydrogenase reactions produce 9 ATP – 1 FADH 2 reaction creates 2 ATP – Succinyl CoA synthetase reaction produces 1 ATP

9.6 Regulation Two levels of regulation – Supply of substrate – Intrinsic activity

9.6 Regulation Supply of substrate – Acetyl CoA – can regulate – Acetyl CoA can come from carbohydrates, fats and proteins – Regulated by phosphorylation and dephosphorylation of E1 component Catalyzed by protein kinases and phosphatase

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9.6 Regulation Intrinsic acitivity – Several steps are metabolically irreversible Citrate synthase Isocitrate dehydrogenase 2-ketoglutarate dehydrogenase Succinyl CoA synthetase Succinate dehydrogenase – All of the above steps are regulated by Calcium

9.6 Regulation Citrate levels can affect glycolysis – Overall increase in flux of Krebs cycle increases citrate – Citrate can be transported across inner mitochondria membrane – Decreases phosphofructokinase activity and glycolysis

9.7 Krebs Cycle as a Second Crossroad of Metabolic Pathways Can be viewed as a metabolic hub Fatty acid synthesis uses citrate Some amino acids can be converted to intermediates – Transamination Heme biosynthesis uses succinyl CoA Glyoxylate cycle in bacteria and plants