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Antioxidant Enzymes Maria Holmstrom Qiang Zhang Nicole Milkovic Erin Rosenbaugh.

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Presentation on theme: "Antioxidant Enzymes Maria Holmstrom Qiang Zhang Nicole Milkovic Erin Rosenbaugh."— Presentation transcript:

1 Antioxidant Enzymes Maria Holmstrom Qiang Zhang Nicole Milkovic Erin Rosenbaugh

2 Beal, Nature. 2006 Oct 19;443(7113):787-95. Introduction to Antioxidant Enzymes

3 Superoxide dismutases Catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide – Diffusion limited Found in nearly all oxygen-exposed cells Categorized by metal prosthetic group – Cu/Zn, Mn, Fe or Ni

4 Species B. SubtilisMnSOD (sodA) E. ColiMnSOD (sodA) and FeSOD (sodB) S. CerevisiaeCuZnSOD (sod1) and MnSOD (sod2) H. SapiensCuZnSOD (sod1), MnSOD (sod2) and EC-SOD (CuZn, sod3)

5 Localization Bacteria – SOD A cytoplasm – SOD B cytoplasm Eukaryotes – SOD1 cytosol – SOD2 mitochondrial matrix – SOD3 (humans) glycated and secreted into the extracellular space, and subsequently anchored to heparan sulfate proteoglycans

6 Catalytic site, bovine SOD1 Image from: Pelmenschikov & Siegbahn, Inorg. Chem, 2005

7 M (n+1)+ − SOD + O 2 − → M n+ − SOD + O 2 M n+ − SOD + O 2 − + 2H + → M (n+1)+ − SOD + H 2 O 2

8 Catalase Catalase is a common enzyme found in nearly all living organisms that are exposed to oxygen, where it functions to catalyze the decomposition of hydrogen peroxide to water and oxygen First noticed by Louis Jacques Thénard in 1818Louis Jacques Thénard First named as catalase by Oscar Loew in 1900Oscar Loew Catalase is a tetramer highest turnover numbers

9 Cofactors Heme Manganese

10 Distribution among organisms All known animals use catalase in every organ, with particularly high concentrations occurring in the liver Catalase is also universal among plants, and many fungi are also high producers of the enzyme Catalase has also been observed in some anaerobic microorganisms

11 Reconstructed phylogenetic tree of 70 typical catalases from all main living kingdoms ANTIOXIDANTS & REDOX SIGNALING Volume 10, Number 9, 2008

12 Catalase genes Bacillus subtilis: katA(vegetative catalase 1), katX(catalase in spores), katE(catalase 2) E. Coli: katE(HPII(III)), katG(HPI), katP(EHEC- catalase) S. Cerevisiae: CTA1(Catalase A), CTT1(Ctt1p ) H. Sapiens: CAT(Catalase)

13 Location Intracellular Extracellular Cell surface Periplasm Cytoplasm Cytosol Glyoxysome Mitochondrion

14 Introduction to Peroxiredoxin Widely distributed thiol-based group of enzymes that catalyze the reduction of H 2 O 2, organic hydroperoxides (ROOH), and peroxynitrite – ROOH +2e-  ROH + H 2 O 3 Classes: Typical 2-Cys, Atypical 2-Cys, 1-Cys

15 Isoforms of Mammalian Peroxiredoxins Wood ZA et al. (2003) Structure, mechanism and regulation of peroxiredoxins. TRENDS Bio Sci 28:32-40

16 Peroxiredoxin Mechanism Wood ZA et al. (2003) Structure, mechanism and regulation of peroxiredoxins. TRENDS Bio Sci 28:32-40

17 1.65 Å Structure Of Prx From Aeropyrum pernix K1 Complexed With H 2 O 2 Nakamura Tet al. (2010) Crystal structure of peroxiredoxin from Aeropyrum pernix K1 complexed with its substrate, hydrogen peroxide. J. Biochem. 147:109-115

18 Journal of Biochemistry. Gordon C. Mills, 1957 Glutathione (GSH) and the Glutathione Peroxidase (GPx) Activity of an Erythrocyte Factor Protect Hemoglobin from Oxidative Breakdown A. Effect of azide (catalase inhibitor) and GSH (reduced glutathione) on the coupled oxidation of hemoglobin by ascorbic acid (AA) B. Concentration-dependent effects of erythrocyte enzyme preparation on choleglobin formation AB Erythrocyte hemosylate containing hemogloblin Crystalline hemogloblin Boiled Enzyme Crystalline hemogloblin + AA + GSH + NaN 3

19 Function of Glutathione Peroxidase (GPx) GPx and GSH remove intracellular hydrogen peroxide and hydroperoxides to protect cellular components from oxidative damage/modifications GPX reduces many reactive oxygen species (e.g., lipid hydroperoxides (ROOR’) to alcohols and to reduce free hydrogen peroxide to water) Glutathione system often functions in parallel with thioredoxin system to regulate the redox homeostasis in cells ROOR' (lipid hydroperoxidase) + electron donor (2 e - ) + 2H + ROH + R'OH 2 GSH (reduced glutathione) + H 2 O 2 GSSG (oxidized glutathione) + 2 H 2 O GPx GPx4

20 Mechanism of Glutathione Peroxidase 2 GSH (reduced glutathione) + H 2 O 2 GSSG (oxidized glutathione) + 2 H 2 O GPx Active Site of Gpx Prabhakar, R. et al. Biochemistry, 2005 Mechanism for GPx catalytic cycle (1) Peroxide (e.g., H 2 0 2 ) reduction and oxidation of the selenolate anion/ selenol (E-Se - or E-Se-H) to selenenic acid (E-SeOH) (2) Selenenic acid reacts with GSH to produce seleno-sulfide adduct (E-Se-SG) (3) 2 nd GSH molecule attacks E-Se-SG to regenerate active GPx and GSSG Selenocysteine residue Reaction 3 is rate limiting step

21 Subcellular Localization of GPx in Mammalian Cells Modified from geneticssuite.net/node/11 GPx1, Gpx2, GPx4 GPx4 Extracellular fluids: GPx3 cGPX or GPX1- cytosol giGPX or GPX2- cytosol, vesicular structures (external cell surface?) Glycosylated GPX, pGPX or GPX3- extracellular, compartments (e.g., plasma) PHGPX or GPX4- mitochondrial membranes, nucleus, nucleolus, cytosol

22 Species and Tissues that express Glutathione Peroxidase GPX1- found in nearly all tissues GPX2- gastrointestinal tract GPX3- extracelluar fluids and low levels in plasma; mRNA predominately in kidney GPX4- ubiquitously expressed; membrane fractions of testis Margis R. et al. FEBS Journal. 2008; GPx gene clusters from Group I → metazoans (animal kingdom) Group II → fungi, proteobacteria, cyanobacteria, algae Group III → plant kingdom

23 Beal, Nature. 2006 Oct 19;443(7113):787-95. Antioxidant Enzymes


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