Glycolysis Can be aerobic or anaerobic Liver and pancreas. Major regulation of glucose in blood Exergonic Discovered by Buchners in 1897. Demonstrated that fermentation could take place outside living cells 1940- glycolytic pathways elucidated by Emben-Meyerhof Starch digested by pancreatic enzyme alpha-amylase. Cleaves alpha 1,4 bonds. NOT the 1,6 bonds. In mammals glucose is only fuel used by the brain Glycolysis is an energy-conversion pathway in many organisms glycolysis takes place in the cytoplasm Stage 1: Glucose Fructose 1,6-bisphosphate 1. Phosphorylation 2. Isomerization 3. Phosphorylation Stage 2: Fructose 1,6-bisphosphate 2 3 carbon compounds Stage 3: ATP harvested when 3 carbon compound oxidized to pyruvate Hexokinase traps glucose in the cell and begins glycolysis Glucose is phosphorylated by ATP to form glucose 6-phosphate. Glucose 6-phosphate cannot pass through the membrane. Destabilizes glucose. Negatively regulated by Glucose 6-phosphate Catalyzed by hexokinase. Requires Mg 2+. Induced fit enzyme Kinases transfer phosphoryl group from ATP to acceptor. Substrate induced cleft closing is a mechanism of all kinases Isomerization of glucose 6-phosphate to fructose 6-phosphate Aldo to a keto Catalyzed by phosphoglucose isomerase Fructose 6-phosphate phosphorylated by ATP to form fructose 1,6-bisphosphate. Phosphofructokinase. Allosteric. High ATP, down regulated. Citrate and glucagon are regulators. F 26 BP activates along with insulin. Fructose 1,6-bisphosphate to glyceraldehyde 3.phosphate and dihydroxyacetone phosphate Catalyzed by aldolase Conversion of glyceraldehyde 3-phosphate into 1,3-bisphosphoglycerate Catalyzed by glyceraldehyde 3-phosphate dehydrogenase. Must be coupled Thioester intermediate 2 ATP 3-phosphoglycerate into pyruvate 2 ATP. Negative: alanine, glucagon Positive: f 1,6-bp, insulin Overall reaction: Glucose + 2 pi + 2 ADP + 2 NAD+ 2 pyruvate + 2 ATP + 2 NADH + 2 H + 2 H20 -96 kj/mol total yield: 2 pyruvate, 2 ATP, 2 NADH Inhibitors of glycolysis Arsenate Fluoride- affects enolase Streptococcus mutans Thrive on sugars, cause dental carries Fluoride inhibits their enolase Other fuels in Glycolysis Galactose, fructose, glycerol Galactose Enters glycolytic pathway as glucose 6-phosphate Lactose + H20 glucose + galactose Galactose is a C4 epimer of glucose Hydrolyzed from lactose by lactase ( enterocytes, small intestine) and beta-galactoside (gut bacteria) Catabolism 1st step is phosphorylation Galactose galactose 1-phosphate Catalyzed by galactokinase Requires ATP Epimerize UDP galactose to UDP glucose to start cycle over again. UDP galactose 4-epimerase Glucose-1phosphate glucose 6-phosphate Catalyzed by phosphoglucomutase Galactosemia High concentration of galactose in system Infant screening Symptoms: irritability, cataracts, brain damage, kidney damage, increase in liver size Causes: loss or reduction of galactokinase, galactose 1-phosphate uridyl transferase, UDP glucose epimerase. Fructose In liver and muscle Fructose?> fructose 1-phosphate Catalyzed by fructokinase Requires ATP Fructose 1-phosphate glyceraldehyde + dihydroxyacetone phosphate Catalyzed by fructose 1-phosphotase Glyceraldehyde glyceraldehyde 3-phosphate Requires ATP Catalyzed by triose kinase In adipose Fructose fructose 6-phosphate Catalyzed by hexokinase Glycerol Breakdown of triacylglycerol enters at stage 3 Glycerol glycerol phosphate dihydroxyacetone phosphate Catalyzed by glycerol kinase and glycerol phosphate dehydrogenase Cori Cycle In liver Gluconeogenesis Lactate pyruvate glucose In muscle Glycolysis Glucose pyruvate lactate Gluconeogenesis Daily brain glucose requirement ? 120 g Daily whole body glucose requirement- 160 g Glucose present in bodily fluids- 20 g Glucose readily available from glycogen ? 190 g The gluconeogenesis pathway converts pyruvate into glucose Noncarbohydrate precursors pyruvate or enter later Major noncarbohydrate precursors are lactate, amino acids, glycerol Lactate- formed in skeletal muscles. Converted into pyruvate by lactate dehydrogenase Major site for gluconeogensis is the liver and a small amount in the kidney Mitochondria Outer membrane Permeable to small molecules by porins Permeable to most anions Intermembrane space Protons pumped in by electron transport chain Innermembrane Folds called cristae. Impermeable to ions and polar molecules Matrix Most enzymes of TCA cycle, Beta oxidation, DNA and RNA Translocators- move ATP/ADP ATP ? ADP Malate ? phosphate Citrate?Malate OH- --- pyruvate OH- --phosphate Gluconeogensis is not a reversal of glycolysis PEP is formed from pyruvate by oxaloacetate Pyruvate pyruvate carboxylase (biotin. Prosthetic group carries C02. Needs acetyl CoA) Oxaloacete formed, made into malate to cross mitochondrial matrix then back into oxaloacetete. Oxaloacete has no transporter 6 high transfer potential phosphoryl groups are spent in synthesizing glucose from pyruvate. Gluconeogensis and glycolysis are reciprocally regulated one is active while the other is inactive when energy is needed, glycolysis will dominate when surplus of energy, gluconeogenesis will take over Glycolysis regulators affecting PFK F-2,6BP + AMP + ATP ? Citrate ? H+ - Gluconeogenesis regulators affecting F16BPtase F-2,6BP ? AMP ? Citrate + Glycolysis regulators affecting pyruvate kinase F1,6-BP + ATP ? Alanine ? Gluconeogenesis regulators affecting pryuvate carboxylase and PEP carboxykinase Acetyl CoA + ADP ? Pyruvate Dehydrogenase Links glycolysis to the citric acid cycle In mitochondrial matrix, pyruvate oxidatively decarboxylated by pyruvate dehydrogenase complex to form acetyl CoA Pyruvate + CoA + NAD Acetyl CoA + CO2+ NADH Irreversible reaction Strategy behind making acetyl coa The thioester bond is less stable than ester bonds so it is energetically favored to transfer the acetyl group to CoA to oxaloacetate to form 6C citrate The synthesis of Acetyl CoA from pyruvate requires three enzymes and five coenzymes Pyruvate dehydrogenase complex Pyruvate dehydrogenase (E1) TPP. Oxidative decarboxylation of pyruvate Dihydrolipoyl transacetylse (E2). Lipoamide. Transfer of acetyl group to CoA Dihydrolipoyl dehydrogenase (E3) Core. FAD. Regeneration of oxidized lipoamide Coenzymes: Thiamin pyrophosphate (TPP), lipoic acid, FAD (these three serve as catalytic cofactors) CoA and NAD+ (stoichiometric cofactors) Pyruvate + Carbanion of TPP - CO2 + Hydroxyethyl-TPP. Catalyzed by pyruvate dehydrogenase component Hydroxyethyl-TPP + Lipoamide carbanion of TPP + Acetyllipoamide (thioester bond) Coenzyme A +acetyllipoamide acetyl CoA + Dihydrolipoamide. Catalyzed by dihydrolipol transacetylase Flexible linkages allow lipoamide to move between different active sites The structural integration of three kinds of enzymes and the long flexible lipoamide arm makes the coordinated catalysis of a complex reaction possible. The proximity of one enzymes to another increases the overall reaction rate and minimizes side reactions BeriBeri I cant I cant Thiamine deficiency. Affects E1. Limited energy The Citric Acid Cycle A citrate synthase forms citrate from oxaloacetate and acetyl CoA Oxaloacetate + acetyl CoA citryl CoA Citrate. Loss of water at citryl intermediate. Adol condensation followed by hydrolysis Catalyzed by citrate synthase Citryl CoA has thioester. Powers synthesis Citrate synthase prevents undesirable reactions Oxaloacetate induces a major structural rearrangement leading to the creation of a binding site for acetyl CoA Open to closed Induced fit Citrate is isomerized into isocitrate Citrate cis-aconitate isocitrate Catalyzed by aconitase (iron-sulfur or non-heme protein) Roles of citrate Intermediate in TCA Activator of fatty acid synthesis Precursor of cytoplasmic acetyl coa Inhibitor of PFK The more citrate, more storage synthesis Isocitrate is oxidized and decarboxylated to alpha-ketoglutarate Isocitrate oxalosuccinate alpha-ketoglutarate Catalyzed by isocitrate dehydrogenase Rate is important NADH and CO2 generated Rate limiting +: ADP, NAD, Mg - : NADH, ATP Succinyl CoA is formed by the oxidative decarboxylation of alpha-ketoglutarate Alpha-ketoglutarate succinyl CoA CO2 and NADH2 formed Alpha-ketoglutarate dehydrogenase complex Negative regulation : ATP, NADH, succinyl coA A compound with high phosphoryl transfer potential is generated from succinyl CoA Succinyl CoA is energy rich thioester compound Succinyl CoA + Pi + GDP succinate + CoA + GTP Formation of GTP at expense of succinyl CoA is substrate level phosphoryaltion. Oxaloacetate is regenerated by oxidation of succinate Succinate fumerate malate oxaloacetate Enzymes: succinate dehydrogenase (iron sulfur protein. Directly associated with oxidative phosphorylaton), fumarase, malate dehydrogenase Generation of FADH2 and NADH The Citric Acid Cycle produces high transfer potential electrons, GTP and CO2 Acetyl CoA + 3 NAD+ + FAD + GDP + Pi+ 2 H2O 2 CO2 + 3 NADH + FADH2 + GTP + 2 H + CoA Entry into the cycle and metabolism through it are controlled The pyruvate dehydrogenase complex is regulated allosterically and by reversible phosphorylation Pyruvate acetyl CoA is irreversible Acetyl CoA inhibits E2 by direct binding NADH inhibits E3 Phosphorylation of pyruvate dehydrogenase component (E1) by a specific kinase switches off complex activity. Deactivation reversed by action of specific phosphotase (stimulated by Ca2+) The Citric Acid Cycle is controlled at several points Primary control points Isocitrate dehydrogenase (ATP inhibited) Alpha-ketoglutarate dehydrogenase (succinyl CoA and NADH inhibited) In bacteria, synthesis of citrate is important control point The citric acid cycle is a source of biosynthetic precursors Citric acid cycle must be capable of being rapidly replenished Oxaloacetate formed by carboxylation of pyruvate Pyruvate carboxylase Anaplerotic reaction Disruption of pyruvate metabolism is cause of beriberi and poisoning by mercury and arsenic Beriberi Cardiovascular and neurological disorder. Thiamine deficiency. Far East. Alcoholics. Pain in limbs, weakness, distorted skin sensation. Heart enlarged. Thiamine is precursor to cofactor thiamine pyrophosphate. Cofactor of pyruvate dehydrogenase, alpha-ketoglutated, transkeotlose Similar symptoms in mercury or arsenic poisoning Binds to E3 Treatment is administration of sulfhydryl reagents with sulfhydryl groups. 2,3-dimercaptopropanol Glyoxylate cycle enables plants and bacteria to grow on acetate Acetyl CoA can be synthesizes from acetate Acetate + CoASH acetyl coa Catalyzed by acetyl coA synthetases Done in gyoxysome organelle Other metabolites Citrate fatty acids, sterols Alpha-ketoglutarate glutamate, other amino acids, purines Succinyl CoA porphyrins, heme, chlorophyll Oxaloacetate aspartate, other amino acids, purines, pyrimidines ATP down, acetyl CoA up, oxaloacetate formed ATP up, acetyl CoA up, oxaloacetate converted to glucose Mammals cannot use acetyl CoA for net conversion to oxaloacetate Oxidative Phosphorylation Each ATP molecule is recycled 300 times a day Oxidative phosphorylation couples the oxidation of carbon fuels to ATP synthesis with a proton gradient Conversion of electron motive force into electron driven pumps. NADH-Q oxidoreductase, Q-cytochrome, c oxidoreductase, cytochrome c oxidase. Large transmembrane complexes Contain quinones, flavins, iron-sulfur clusters, heme, copper ions Oxidative phosphorylation in eukaryotes takes place in the mitochondria Mitochondria contain the respiratory assembly, enzymes of the TCA and enzymes of fatty acid oxidation Mitochondria are bound by a double membrane Inner membrane folded into cristae Two compartments Intermembrane space Matrix Oxidative phosphorylation takes place on inner membrane Outer membrane is permeable to small molecules and ions Porins VDAC- voltage dependent anion channel Regulated flux of metabolites across outer membrane Inner membrane impermeable to all ions and polar molecules Lots of transporters N side = inner (matrix) negative charge P side = outer (cytoplasm) positive charge Mitochondria are the result of an Endosymbiotic event Oxidative phosphorylation depends on electron transfer Highly exergonic reduction of oxygen to water Electron transfer potential of an electron is measured as a redox potential Phosphoryl transfer potential = delta G zero prime Electron transfer potential+ dealt E prime zero Negative reduction potential means oxidized form of a substance as a lower affinity for electrons than H2 A strong reducing agent (NADH) is poised to donate electrons and has a negative reduction potential A strong oxidizing agent (O2) has positive reduction potential A 1.14 volt potential difference between NADH and molecule oxygen drives electron transport through the chain and favors the formation of a proton gradient The respiratory chain consists of four complexes, three proton pumps and a physical link to the TCA NADH-Q oxidoreductase ? complex I Accepts electrons from NADH Passes electrons to ubiquinone Pumps protons into intermembrane space NADH dehydrogenase Electron carrier: FMN, Fe-S centers Succinate-Q reductase- complex II Carriers: FAD, Fe-S centers Accepts electrons from FADH2 Passes electrons to ubiquinone to form ubiquinol Does NOT pump protons into intermembrane space Q-cytochrome c oxidoreductase- complex III Electron carriers: 3 cytochromes, Reiske Fe-S Q-cytochrome c oxidoreductase Accepts electrons from ubiquinol Passes electrons to cytochome c Pumps protons into intermembrane space Cytochrome c oxidase- complex IV Carriers: cytochrome A Cytochrome c oxidase Accepts electrons from cytochome c Passes electrons to oxygen Pumps protons into intermembrane space I, III, IV are associated with supramolecular complex respirasome Mobile electron carriers Ubiquinone Co enzyme Q Hydrophobic, travels within the inner membrane Accepts electrons from complex I, II, glycerol 3-phosphate shuttle Carries electrons to complex III Cytochrome c Hydrophilic, travels in intermembrane space Accepts electrons from complex III Carries electrons to complex IV NADH NADH-Q oxidoreductase Q (ubiquinone)- Q-cytochrome c oxidoreductase cyt c cytochrome c oxidase O2 The high potential electrons of NADH enter the respiratory chain at NADH-Q oxidoreductase NADH binds to FMN prosthetic group Ubiquinol is the entry point for electrons from FADH2 of flavoproteins Glycerol phosphate dehydrogenase and fatty acyl CoA dehydogenase transfer their high potential electrons from FADH2 to Q to form ubiquinol Electrons flow from ubiquinol to cytochrome C through Q-cytochrome c oxidase Catalyzes electrons from Qh2 to cyt c A cytochrome is an electron transferring protein that contains a heme prosthetic group The two cytochrome subunits of q-cytochrome c oxidoreductase contain heme bl, bh, ci The Q cycle funnels electrons from a two electron carrier to a one electron carrier and pumps protons Cytochome C oxidase catalyzed the reduction of molecular oxygen to water Toxic derivatives of molecule oxygen such as superoxide radical are scavenged by protective enzymes Electrons can be transferred between groups that are not in contact A proton gradient powers the synthesis of ATP ATP synthase Ball and stick Fo contains the proton channel Rotor- moving unit. C ring, gamma stalk Stator- stationary unit Binding change mechanism Beta subunits performs steps 1. ADP and pi binding (loose) ATP synthesis(tense) ATP release(open) rotational catalysis is the worlds smallest molecule motor proton flow around the c ring powers ATP synthesis Many shuttles allow movement across the mitochondrial membrane glycerol 3-phohsphate shuttle reduction of DHAP to glycerol 3-P reduction of FAD to FADH2 reduction of MT electron carrier to Q malate-aspartate shuttle. Heart and liver crossing over Inhibition complex I- blocked by rotenone and amytal complex III- blocked by antimycin A complex IV- blocked by CN, NH3, CO ATP synthase- olgiomycin Uncouplers Another way around ATP synthase UCP-1 (thermogenin) Provides direct access to H+ from intermembrane space to matrix. Used to generated heat Brown fat DNP Production of TNT. Poison Glycogen Metabolism Glycogen is the readily mobilized storage form of glucose Linked by alpha-1,4-glycosidic bonds but branches are alpha-1,6 Doesn?t require oxygen More glycogen in the liver than muscle In liver, used to maintain blood glucose levels In muscle, used for energy itself Glycogen metabolism is the regulated release and storage of glucose Glycogen degradation 1. Release of glucose 1-phosphate from glycogen. 2. Remodeling of glycogen substrate 3. Conversion of glucose 1-phosphate to glucose 6-phosphate glucose 6-phosphate can either go into glycolysis (muscle, brain), glucose to the blood (liver) or ribose-NADPH pathway regulation by hormones adjusts glycogen metabolism to meet the needs of the entire organism Glycogen breakdown requires the interplay of several enzymes requires 4 enzyme activities Phosphorylase Glycogen + Pi glucose 1-phosphate + glycogen Removes glucosyl residues from free ?OH ends Reaction is readily reversible Cofactor is pyridoxal phosphate Debranching enzymes Phosphorylase can only cleave alpha-1,4 bonds NOT alpha-1,6 bonds Transferease and alpha-1,6 glucosidase remodel glycogen Transferase- shifts blocks Alpha-1,6 glucosidase hydrolyses Phosphoglucomutase Glucose 6-phosophate Contained in the liver, absent in muscle Glucose 6-phosphate + H20 glucose + Pi Epinephrine and Glucagon signal the need for glycogen breakdown PKA phosphorylase kinase glycogen phosphorylase G proteins transmit the signal for the initiation of glycogen breakdown Muscular activity releases epinephrine. Derived from tyrosine Liver is more responsive to glucagon AMP- signal transduction cascade Epinephrine or glucagon bind to a protein on the membrane. 7TM receptor Activates GDP ( which as alpha, beta and gamma subunits) GTP attaches to Adenylate cyclase which turns on cyclic AMP cascade Glycogen breakdown must be readily turned off when necessary Shut down automatically when no hormone present Glycogen is synthesized and degraded by different pathways Synthesis: glycogen n + UDP-glucose glycogen n +1 + UDP Degradation: glycogen n+1 + Pi glycogen n + glucose 1-phosphate UDP-glucose is an activated form of glucose Glycogen synthase catalyzes the transfer of glucose from UDP-glucose to a growing chain Glycogenin- primer Needs 4 residues already to add on A branching enzyme forms alpha 1,6 linkages Branching increases rate of glycogen synthesis and degradation Glycogen synthase
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