Antioxidant Enzymes

Maria HolmstromQiang Zhang

Nicole MilkovicErin Rosenbaugh

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

Introduction to Antioxidant Enzymes

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

Species

• B. Subtilis MnSOD (sodA)• E. Coli MnSOD (sodA) and FeSOD

(sodB)• S. Cerevisiae CuZnSOD (sod1) and MnSOD

(sod2)• H. Sapiens CuZnSOD (sod1), MnSOD (sod2)

and EC-SOD (CuZn, sod3)

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

Catalytic site, bovine SOD1

Image from: Pelmenschikov & Siegbahn, Inorg. Chem, 2005

Image from: Pelmenschikov & Siegbahn, Inorg. Chem, 2005

M(n+1)+ − SOD + O2− → Mn+ − SOD + O2

Mn+ − SOD + O2− + 2H+ → M(n+1)+ − SOD + H2O2

CatalaseCatalase 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 1818

First named as catalase by Oscar Loew in 1900

Catalase is a tetramer

highest turnover numbers

Cofactors• Heme

• Manganese

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

Reconstructed phylogenetic tree of 70 typical catalases from all main living kingdoms

ANTIOXIDANTS & REDOX SIGNALING Volume 10, Number 9, 2008

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)

Location

• Intracellular• Extracellular• Cell surface• Periplasm• Cytoplasm• Cytosol• Glyoxysome• Mitochondrion

Introduction to Peroxiredoxin

• Widely distributed thiol-based group of enzymes that catalyze the reduction of H2O2, organic hydroperoxides (ROOH), and peroxynitrite– ROOH +2e- ROH + H2O

• 3 Classes: Typical 2-Cys, Atypical 2-Cys, 1-Cys

Isoforms of Mammalian Peroxiredoxins

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

Peroxiredoxin Mechanism

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

1.65Å Structure Of Prx From Aeropyrum pernix K1 Complexed With H2O2

Nakamura Tet al. (2010) Crystal structure of peroxiredoxin from Aeropyrum pernix K1 complexed with its substrate, hydrogen peroxide. J. Biochem. 147:109-115

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

A BErythrocyte hemosylate containing hemogloblin

Crystallinehemogloblin

Boiled Enzyme

Crystalline hemogloblin + AA + GSH + NaN3

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) + H2O2 GSSG (oxidized glutathione) + 2 H2OGPx

GPx4

Mechanism of Glutathione Peroxidase 2 GSH (reduced glutathione) + H2O2 GSSG (oxidized glutathione) + 2 H2O

GPx

Active Site of Gpx

Prabhakar, R. et al. Biochemistry, 2005

Mechanism for GPx catalytic cycle

(1) Peroxide (e.g., H202) 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) 2nd GSH molecule attacks E-Se-SG to regenerate active GPx and GSSG

Selenocysteine residue

Reaction 3 is rate limiting step

Subcellular Localization of GPx in Mammalian Cells

Modified from geneticssuite.net/node/11

GPx1, Gpx2, GPx4

GPx4

GPx4

Extracellular fluids: GPx3cGPX 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

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 fromGroup I → metazoans (animal kingdom)Group II → fungi, proteobacteria, cyanobacteria, algaeGroup III → plant kingdom

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

Antioxidant Enzymes