Pentose Phosphate Pathway Biochemistry PDF

1A BIOCHEMISTRY Pentose Phosphate Pathway BRENDO V. JANDOC, MD OVERVIEW...

1A BIOCHEMISTRY Pentose Phosphate Pathway BRENDO V. JANDOC, MD OVERVIEW FIRST STAGE PENTOSE PHOSPHATE PATHWAY (IRREVERSIBLE OXIDATIVE REACTIONS)  also called Hexose Monophosphate Pathway or  Consists of 3 Reactions and formation of 6-Phosphoglucunate Pathway a. Ribulose 5-phosphate  occurs in the cytosol and in tissues b. CO2 - Liver: fatty acid synthesis c. 2 molecules of NADPH (for each G6P oxidized) - Mammary Glands: fatty acid synthesis  Important in the - Adipose Tissue: fatty acid synthesis a. Liver, Lactating Mammary Glands - Adrenal Cortex: NADPH-dependent synthesis of fatty acid biosynthesis steroids b. Adrenal Cortex - Erythrocyte: require NADPH to keep glutathione NADPH-dependent steroids synthesis reduced c. RBCs  no ATP is directly consumed or produced in the cycle NADPH requirement to keep glutathione  Consists of reduced for membrane integrity a. Irreversible Oxidative Reactions (1st stage of PPP) - oxidation of glucose generates NADPH A. Dehydrogenation of Glucose 6-Phosphate - glucose-6-phosphate undergoes PPP is regulated primarily at this step dehydrogenation and decarboxylation to yield  Irreversible Reaction a pentose, ribulose-5-phosphate G6P oxidation 6-phosphogluconolactone b. Reversible Sugar-Phosphate Interconversions (6-phosphoglucono-δ-lactone) (2nd & 3rd stages of PPP)  Enzyme: glucose 6-phosphate dehydrogenase (G6PD) - functions in several different directions Coenzyme: NADP+ reduced to NADPH - ribulose-5-phosphate is converted back to  NADPH: competitive inhibitor of the enzyme glucose-6-phosphate by a series of reactions high NADPH/NADP+ enzyme activity inhibition involving mainly two enzymes: *Increased NADPH Demand decreased NADPH/NADP+ transketolase and transaldolase increased glucose 6-phosphate dehydrogenase activity increased  Carbon 1 of Glucose 6-Phosphate released as CO2 flux through the cycle  Supply of intermediates and Demand for intermediates  Insulin: enhances glucose 6-phosphate dehydrogenase determine Rate and Direction of the Reactions gene expression flux through the pathway increases in well-fed state B. 6-Phosphogluconolactone Hydrolysis and Ribulose 5-Phosphate Formation Enzymes  6-Phosphogluconolactone Hydrolase/Lactonase - hydrolyze the lactone (cyclic ester) of 6-phosphogluconolactone to 6-phosphogluconate - irreversible reaction and not rate-limiting  6-Phosphogluconate Dehydrogenase - catalyzes oxidative decarboxylation of 6- phosphogluconate by NADP+ FUNCTIONS OF PENTOSE PHOSPHATE PATHWAY - irreversible 1. Source of NADPH Products  produces 2 NADPH for each glucose 6-phosphate  Pentose sugar-phosphate (ribulose 5-phosphate) molecule entering the oxidative part of the pathway  CO2 (from carbon of glucose)  NADPH  2nd molecule of NADPH - reducing equivalent for GSH/GSSG (the premier antioxidant system in the cell) REVERSIBLE OXIDATIVE REACTIONS - steroid and fatty acid biosynthesis  Occurs in all cell types synthesizing nucleotides 2. Source of Ribose 5-Phosphate & nucleic acids - shunts G6P to form ribulose-5-phosphate for  Interconversions of 3-, 4-, 5-, 6-, and 7-carbon sugars nucleotide synthesis  Ribulose 5-Phosphate conversion to 3. Route for Use of Ingested Pentoses a. Ribose 5-Phosphate - conversion to fructose 6-phosphate (F6P) and b. Intermediates of Glycolysis glyceraldehyde 3-phosphate (G3P) - fructose 6-phosphate - glyceraldehyde 3-phosphate  Controlled by the availability of intermediates Trans | 1 of 8  Biochemistry Pentose Phosphate Pathway  TPP (Thiamin Pyrophosphate) THIRD STAGE PENTOSE PHOSPHATE PATHWAY required in the transketolase reaction (REVERSIBLE OXIDATIVE REACTIONS)  Other TPP-Requiring Enzymes 3 five-carbon sugars are converted to 2 fructose-6-phosphate and Pyruvate Decarboxylase: pyruvate dehydrogenase complex 1 glyceraldehyde-3-phosphate α-Ketoglutarate Dehydrogenase: TCA cycle Branched-Chain α-Keto Acid Dehydrogenase: branched- A. Ribose 5-Phosphate and Xylulose 5-Phosphate to chain amino acid metabolism Sedoheptulose 7-Phosphate and G3P  Reaction A. Conversion of Pentose Phosphate to Intermediates of Glycolysis 2-carbon unit is transferred from a ketose (xylulose 5-phosphate) to an aldose (ribose 5-phosphate) seven-carbon sugar cells that carry out reductive biosynthetic reactions sedoheptulose-7-phosphate and glyceraldehyde-3-phosphate  Enzyme: transketolase (requires TPP as cofactor) greater need for NADPH (than for ribose 5-phosphate)  Wernicke-Korsakoff Syndrome - from chronic thiamine deficiency reduced or transketolase (transfers 2-carbon units), defective transketolase activity (due to reduced transaldolase (transfers 3-carbon units) affinity to TPP) clinical manifestations of this syndrome (symptoms: weakness or paralysis, convert ribulose 5-phosphate to impaired mental function) glyceraldehyde 3-phosphate and fructose 6-phosphate B. Sedoheptulose 7-Phosphate and G3P to glycolysis Erythrose 4-Phosphate and F6P  Reaction B. Formation of Ribose 5-Phosphate 3-carbon unit is transferred from sedoheptulose 5-phosphate to G3P from Intermediates of Glycolysis fructose-6-phosphate and the four-carbon sugar erythrose-4-phosphate demand for pentoses (for  Enzyme: transaldolase (requires no cofactor) nucleotide and nucleic acid synthesis) > NADPH need C. Xylulose 5-Phosphate and Erythrose 4-Phosphate to F6P and G3P  Enzyme: TPP-dependent transketolase ribose 5-phosphate synthesis from glyceraldehyde 3-phosphate SUMMARY of REACTIONS (STOICHIOMETRY) and 3G6P + 6NADP+ 2F6P + 3CO2 + G3P + 6NADPH + 6H+ fructose 6-phosphate REGULATION OF THE PATHWAY A. Rate of the Glucose-6-Phosphate Dehydrogenase (G6PD) SECOND STAGE PENTOSE PHOSPHATE PATHWAY Reaction (REVERSIBLE OXIDATIVE REACTIONS)  regulate the flux of glucose-6-phosphate Ribulose 5-Phosphate to Ribose 5-Phosphate and  controlled by the availability of its substrate NADP+ so that Xylulose 5-Phosphate the pathway flux increases in response to increasing levels *by isomerization of ribulose 5-phosphate of NADP+ (which indicates increased cellular demand for Enzymes NADPH)  Phosphopentose Isomerase B. NADP+ (Ribulose-5-Phosphate Isomerase)  cellular concentration is the major factor - converts ribulose 5-phosphate to ribose-5-  availability regulates the rate-limiting G6PD reaction phosphate: can be used to produce nucleosides for RNA and DNA synthesis  Phosphopentose Epimerase USES OF NADPH (Ribulose-5-Phosphate Epimerase) - converts ribulose-5-phosphate to xylulose-5- A. Reductive Biosynthesis phosphate part of the energy of G6P is conserved in NADPH 1. NADPH  high-energy molecule  electrons are used in reductive biosynthesis of macromolecules (production of fatty acids and steroids)  cells keep the [NADP+]/[NADPH] ratio near 0.01 (which favors reductive biosynthesis) S1T1 2 of 8  Biochemistry Pentose Phosphate Pathway  pathways that require NADPH  highly reactive, damage to DNA, proteins, unsaturated DETOXIFICATION lipids cell death - Reduction of Oxidized Glutathione  reactive oxygen intermediates are implicated in - Cytochrome P450 Monooxygenase reperfusion injury, cancer, inflammatory disease, aging REDUCTIVE SYNTHESIS  protective mechanisms of the cell - Fatty acid synthesis - Fatty acid chain elongation - Cholesterol Synthesis - Neurotransmitter Synthesis - Deoxynucleotide Synthesis - Superoxide Synthesis 2. NADH  oxidative metabolism (catabolism)  cells keep the [NAD+]/[NADH] ratio near 1000 (which 2. Enzymes that Catalyze Antioxidant Reactions favors metabolite oxidation) a. Glutathione Peroxidase  selenium-requiring B. Reduction of H2O2  converts reduced glutathione (tripeptide thiol, 1. H2O2 ϒ-glutamylcysteinylglycine) oxidized  one of the reactive oxygen species glutathione glutathione reductase using NADPH as a  formed from partial reduction of molecular O2 (a biradical, source of reducing electrons regeneration of reduced has two anti-bonding electrons with parallel spins; glutathione tendency to form superoxide, nonradical hydrogen  chemically detoxify H2O2 peroxide, and hydroxyl radical) The structure of glutathione. The Formation of reactive intermediates from molecular oxygen sulfhydryl group of glutathione, which is oxidized to a disulfide.  formed continuously - as by-product of aerobic metabolism - through reactions with drugs and environmental toxins - diminished levels of antioxidants  oxidative stress: occurs when the rate of ROS and RNOS production overbalances the rate of their removal by cellular defense mechanisms (enzymes and antioxidants) Glutathione peroxidase transfers electrons from GSH to hydrogen peroxide; reduces hydrogen peroxide to water. Glutathione redox cycle. Glutathione reductase regenerates glutathione. *RBCs  totally dependent on HMP pathway for NADPH supply  glucose 6-phosphate dehydrogenase defect decreased NADPH levels nonreduction of oxidized glutathione H2O2 accumulation membrane instability lysis S1T1 3 of 8  Biochemistry Pentose Phosphate Pathway b. superoxide dismutase c. catalase a. NADPH - provides the reducing equivalents - critical for the liver microsomal cytochrome P450 monooxygenase system b. Overall Reaction Catalyzed by a Cytochrome P450 Enzyme Actions of antioxidant enzymes. G-SH = reduced glutathione; G-S-S-G = oxidized glutathione. Compartmentalization of free radical defenses  Various defenses against ROS are found in the various subcellular compartments of the cell.  Highest activities of these enzymes are found in the liver, adrenal gland, and kidney where mitochondrial and peroxisomal contents are high, and cytochrome P450 enzymes are found in abundance in the smooth muscle endoplasmic reticulum.  Glutathione is a nonenzymatic antioxidant. Cytochrome P450 Monooxygenase Cycle 3. Antioxidant chemicals -detoxify oxygen intermediates -correlated with reduced risk for certain types of cancers reduced frequency of other chronic health problems (clinical trials with antioxidants failed to show clear beneficial effects) a. Ascorbic acid (vitamin c) b. Vitamin e c. β-carotene - dietary supplementation: rate of lung cancer in smokers increased C. Cytochrome P450 Monooxygenase System Functions 2. Mitochondrial Cytochrome P450 Monooxygenase System -major pathway for the hydroxylation of aromatic and aliphatic -in hydroxylation of steroids more water soluble compounds (steroids, alcohol, drugs, other compounds) A. Placenta, ovaries, testes, adrenal cortex -detoxify drugs and other compounds converting to soluble - hormone-producing tissues form renal excretion - hydroxylate intermediates in the conversion of cholesterol to steroid hormones 1. Monooxygenases B. Liver - bile acid synthesis (Mixed Function Oxidases) C. Kidney -to hydroxylate 25-hydroxycholecalciferol (Vitamin D) -incorporate one atom from biologically active 1, 25- hydroxylated form molecular oxygen into a substrate (creating a hydroxyl group) the other atom reduced to water S1T1 4 of 8  Biochemistry Pentose Phosphate Pathway 3. Microsomal Cytochrome P450 Monooxygenase System - with the endoplasmic reticulum membranes in the liver A. Detoxification of foreign compounds (Xenobiotics) -Drugs & Pollutants: petroleum products, carcinogen, pesticides B. Purposes of the modification -Activate or inactivate a drug -Makes toxic compound more soluble excretion in the urine or feces C. New hydroxyl group -site for conjugation with a polar compound (glucuronic acid) increased solubility D. Phagocytosis by WBCs (Neutrophils, Macrophages, Monocytes) 1. Phagocytosis -ingestion by receptor-mediated endocytosis of microorganisms, Production of Reactive Oxygen Species during the phagocytic burst foreign particles, cellular debris by activated neutrophils 2. Bacterial Killing Mechanisms a. activation of NADPH oxidase- initiates respiratory burst + a. Oxygen-Dependent Mechanisms superoxide > plasma membrane invaginates, superoxide released -Myeloperoxidase (MPO) System: most potent of the bactericidal into vacuole space mechanisms b. Superoxide > H2O2 -Other Systems: involving the generation of oxygen-derived free c. Myeloperoxidase > HOCl and other halides radicals d. H2O2 > hydroxyl radical from Fenton reaction b. Oxygen-Independent Systems e. iNOS activated > NO -utilize pH changes in the phagolysosome and lysosomal enzymes f. NO + superoide > peroxynitrite > RNOS 3. Mechanism 4. NADPH Oxidase invading bacterium > - hormonally regulated complex enzyme recognized by the immune - subunits contain cytochrome & flavin coenzymes system > attacked by -electrons move from NADPH to O2 via FAD and heme to antibodies > binding to a generate O2 receptor on a phagocytic cell >phagocytosis > 5. Genetic Deficiencies of NADPH Oxidase internalization of the Chronic Granulomatosis microorganism >NADPH - severe, persistent chronic pyogenic infections oxidase (WBC cell - formation of granulomas (nodular areas of inflammation) that membrane) > conversion of sequester the bacteria that were not destroyed molecular O2 to superoxide (respiratory burst -rapid E. Synthesis of Nitric Oxide oxygen consumption - a free radical gas accompanying superoxide -as a mediator in a broad array of biologic systems formation) > converted to -endothelium-derived relaxing factor, which causes vasodilation H2O2 by superoxide by relaxing vascular smooth muscle dismutase (SOD) > addition of -acts as a neurotransmitter chloride ions >hypochlorous -prevents platelet aggregation acid (HOCl) formation > -plays an essential role in macrophage function bacterial killing 1. Synthesis *Excess H2O2 is neutralized by -Substrates: Arginine, O2, and NADPH catalase and glutathione -Coenzyems: Flavin mononucleotide (FMN), flavin adenine peroxidase dinucleotide (FAD), heme, and tetrahydrobiopterin -Products: NO and citrulline -3 no synthases have been identified. Two are constitutive (synthesized at a constant rate regardless of physiologic demand), Ca2+-calmodulin dependent enzymes- found primarily in endothelium (eNOS), and neural tissue (nNOS), and constantly produce low levels of NO. S1T1 5 of 8  Biochemistry Pentose Phosphate Pathway - additional membrane protein oxidation > rigid and nondeformable -An inducible, Ca2+-calmodulin independent enzymes rbcs > removed from the circulation by macrophages (liver, spleen) (iNOS) > hepatocytes, macrophages, - All cells are affected but most severe in RBCS Monocytes, and neutrophils PPP provides the only means of NADPH generation in RBCS -Tumor necrosis factor-α, bacterial endotoxins, and RBCS are devoid of nucleus and ribosomes > cannot renew inflammatory cyto kines > promote synthesis of iNOS, > large supply of enzymes amounts of NO being produced over hours or even days other tissues have alternative sources of NADPH (NADP+ dependent malate dehydrogenases) 2. Actions of NO on Vascular Endothelium -important mediator in the control of vascular smooth muscle B. Precipitating Factors in G6PD Deficiency -synthesized by eNOS in endothelial cells, and diffuses to vascular - Most individuals do not show clinical manifestations smooth muscle > activates the cytosolic form of Guanylate Cyclase - some patients with G6PD deficiency develop Hemolytic Anemia if (also known as guanylyl cyclase) to form cGMP > rise in cGMP > they are treated with oxidant drugs, ingest fava beans, or contract activation of protein kinase G, which phosphorylates Ca2+ channels a severe infection > decreased entry of Ca2+ into smooth muscle cells >decreases the calcium-calmodulin activation of myosin light-chain Kinase > smooth 1. Oxidant Drugs muscle relaxation A. Antibiotics: Sulfamethoxazole & Chloramphenicol -Vasodilator nitrates: nitroglycerin and nitroprusside B. Antimalarials: Primaquine >metabolized to nitric oxide > relaxation vascular smooth muscle C. Antipyretics: Acetanilid &Aspirin > lowers blood pressure. D. Nitrofurans -Sildenafil citrate > treatment of erectile dysfunction, 2. Favism inhibits the phosphodiesterase that inactivates cGMP - G6PD deficiency exacerbated (hemolytic effect) by consumption of fava beans usually within 24-48 hours after consumption 3. Role of NO in mediating macrophage bactericidal activity: - not observed in all patients -iNOS activity is normally low, stimulated by bacterial - all patients with favism have G6PD deficiency lipopolysaccharide and γ-interferon release in response to - mediterranean variant of G6PD deficiency is particularly infection susceptible -Activated macrophages form superoxide radicals that combine -Fava Bean: dietary staple in Mediterranean region with NO to form intermediates that decompose, forming the highly bactericidal OH radical 3. Infection - Most common precipitating factor of hemolysis in G6PD deficiency - infection > inflammatory response > generation of free radicals in G6PD DEFICIENCY macrophages > diffuse into RBCs > oxidative damage - Most common disease-producing abnormality in humans - Highest prevalence in the Middle East, Tropical Africa, Asia, & parts 4. Neonatal Jaundice of the Mediterranean - From impaired hepatic catabolism or increased bilirubin production - inherited disease (X-linked) characterized by Hemolytic Anemia - 1-4 days after birth due to inability to detoxify oxidizing agents - may be severe - family of deficiencies caused by >400 different mutations in the gene coding for G6PD C. Properties of the Variant Enzyme - some cause clinical symptoms 1. Some mutations - shortened life span of patient due to complications of from chronic - do not disrupt the structure of the enzyme’s active site > hemolysis no effect on enzymatic activity - increased resistance to Falciparum Malaria 2. Mutant enzymes - show altered kinetic properties A. Role of G6PD in RBCs A. Decreased catalytic activity Decreased G6PD activity > impaired NADPH formation increases the B. Decreased stability sensitivity of red blood cells to oxidative stress > failure to maintain C. Alteration of binding affinity for NADP+, NADPH, or Glucose 6- adequate amount of reduced glutathione > impaired Phosphate detoxification of free radicals and peroxides > diminished stability > 3. Severity of the disease Hemolysis - correlates with the amount of residual enzyme activity in the RBCs 4. G6PD A- 1. Glutathione - prototype of the moderate (class III) form of the disease -maintain the reduced state of sulfhydryl groups of proteins - RBCs contain unstable but kinetically normal G6PD with most of the (hemoglobin) enzyme activity present in the reticulocytes and younger RBCs - oxidation of sulfhydryl groups > protein denaturation > form (oldest RBCs contain the lowest level of enzyme activity > insoluble masses (Heinz bodies) > attach to RBC membranes preferentially removed in hemolytic episode) S1T1 6 of 8  Biochemistry Pentose Phosphate Pathway 5. G6PD Mediterranean - prototype of the more severe (class II) deficiency - enzyme shows normal stability but scarcely detectable activity 6. Class I Mutations - often associated with Chronic Nonspherocytic Anemia (occurs even in the absence of oxidative stress) Hemolysis Caused by ROS 1. ATP AND NADH –integrity of membrane 2. NADPH from PPP 3. NADPH for reduction of GSSH (oxidized glutathione) to GSH (reduced glutathione) 4. glutathione defense system is compromised in G6PD Deficiency, infections, certain drugs, and purine glycosides of fava beans 5. Heinz bodies, aggregates of cross-linked hemoglobin > mechanical stress as it pass through capillaries plus ROS on membrane > hemolysis D. Molecular Biology of G6PD 1. Mutations - cloning and G6PD gene and sequencing of its complimentary DNA > identification of mutations (most are missense point mutations in the coding region of the gene) that cause G6PD deficiency A. G6PD A-, G6PD Mediterranean - have mutant enzymes by a single amino acid B. Locations I. Clustered near the carboxyl ends of the enzyme - cause Nonspherocytic Hemolytic Anemia II. Amino end of the enzyme - cause milder forms References:  Harvey RA. Lippincott’s Illustrated Reviews: Biochemistry. 5th ed. Philadelphia: Lippincott Williams & Wilkins, 2011.  Dr. Jandoc’s hand-out  S1T1 7 of 8 Biochemistry Pentose Phosphate Pathway S1T1 8 of 8