Oxygen'98Sunrlse Ftee Radical Schoo|November 20-28, lggaWashington D.C.'Naturally Occurring Antioxidants"ANTIOXIDANT ENZYMES AND FUNCTIONSGarry R. Buettncr, ph. D.Free Radical Research lnstiruteEMRB 68The University of lowaIowa City, IA 52242-IlOlTel: 319/335-6749FAX: 319/33b-9049email: gaq/[email protected] BuettnerAntioddant Enzyn€sB.IIIITIIIIIIIA[tioxidatrt Erzvmes and FunctionI. Superoaide Dismutaae (SOD) , 1968-1969, Mccord and Fridovich (JBC)A, Functlo!SODo2'- + o2'- + 2H* e H2o2 + oz (lq.6ryd" 2-4 x loe M's-')Only enzlrrte known to act on a radical.Revolutionary idear the prcsence of SOD implies O2'_ produced in cell during normalmetabolism,*Note - SOD is a prtasry antioxidant enzyme - acts on a ROSWhat is unique about SOD?Teblc 1 Procrryotlc CclL - gODMW/Da SubunitsFeSOD 40,000 2 dimerMnSOD 40,000 2 dimcr80,000 4 tetramerFothiDifferences:1. a.a. sequence2. active metal aite3. cellular locationTablc 2 Eucrryotlc CeI! - SODMW/Da SubunitsMnSOD 88,000 4CUZDSOD 32,000 2Ec (cuzn) SOD 13s,0oo 4EC MnSOD 150,000 2,4EC = exEaceuular .ba4--t h!rE6 ud l\scdo.G.R, Bucttlct Pape IGR BuettnerAntloidant EnzymesC. Iltracellul.r Locatlon of SOD'3a. ProcaiYotes:MnSOD - matrix (innerlFeSOD - outer membraneIIIIIIItIIIIIb. Eucaryotes:CuznSOD - cytoplasm, nucleus, lysosomesMnSOD - mitochondrial matrixEC(CuZn) SOD - Plasma membrane, extracellularECMnSOD - plasrna membraneD, Structurc a!.l PtoPcttlGaa. CUZnSODLargely, acidic Proteins PI 4_6 150 _ 155 a a residues per SOD subunit- Mw = 32,000 Da, dimerhigh Blycine, low tyrosine & tryptophanStability* One of the most stable ProteinsNot dissociated by SDS alone (breaks apart H bonds)Disassociated by: SDS + p-mercaptoethanol or EDTA + heat 40-55"CT1/2 = temperature to break apart 5070 in 10 min - 67'CActivity is constant from pH 4 5 to 9.5Stable to repeated freeze thaw cycles and to Prolonged refrigeration'Inactivated by:6 M guanidine hYdrochloride1-5 mM NacN (cyanide binds coPPer)Andond.rt Esto.. Ed Fuctro!G.R. Bucttncr PaSc IGR BueltnerAnUondant EnztmesIIIIIIIITItIIIIIIIt1-5 mM DDC, diethyldithiocarbamate (binds copper)(roN-C_J 's_nNaCN and DDC are used to inhibit CuZnSOD, but do not aliect MnSODb. qD Cryrtr.tllric Structurc of CuZtrSOD froE Bovin€ ErythrocytcaCu and Zn are 6 A apart2 Cu on separate strands are 34 A apartZn binds 3 His arld I Asp (His 61, His 69, His 78, Asp 81)Cu binds 4 His - active site (His 44, His 46, His 61, His 1181lmidazole of His 6l lies bctween Cu and ZnRepresentative ofpob?eptide structure of bovine CuZn SODOberley, L-W. Superoxide Dismutase, Vol. I, p. 28, 1982-t1.-a-A!tlond.!t E!ztE.. ud F6ctlo!G.R. BucttDcr Pa€e 3GR BuettnerAnUolddant Enzymesc. Chemical Modifications of CUZnSODH2O2 - limits actiYityAt low concentrations change cupric (Cu2+) io cuprous (Cul+) and causereversible inacrivarionHigh concentlations or loog exposures cause irreversible inactivation.Butanedione and phenylglyoxal inactivate CuZnSOD by modifying arginineresidues. Arg 141 iD acitve sited. Catahtic Mechanism lE = enzyme)E-Cu2+ + O2'- -+ E-Cu+l + 02 electron transferE-Cul+ + O2'' + 2H+ J E-cu2+ + H2o2 proton and e- transferBridging Imidazolate H,?othesis - A is correcrIIIIIIIIIIIIItltItlIAr\l-Ctr':1.,I-N\./NHI r-------1 \ /l)+ | ^ | \/')+-Zn--N -.: j_N -Cu--r-t:l\ -F' *t1'andond&t Elzyn . ud Futctlo! C.R. BucttDcr Pase 4FhIFIIIttIGR Buettnertultioddant DM- mese. ECSOD - Marklatd, 1982slighdy hydrophobic gtycoprcteinMW = 135,000 Da4 equal, noncovalently bound subunits4 Ctt and 4 Z^Inhibited by cyanide, azide, H2O2, DDC, SDSThree fiactions, according to binding of heparinSepharose: A, no allinity; B, weak affinity; C, high ajfinity.240 amino acids per subunit; 18 a.a. are signal pepiideMW = 24,174 DaN-glycosylation site {Asn 89)First 95 a.a_ show !o sequence homologies with CuZnSODFrom His 96 to cly 193, ECSOD has strong homolog/ to CuZnSOD_ 49 ol 76 positions areidenticai.ECSOD shares amino acids in 22 of 23 positions in which CuZn is invariant.AllligandstoCu(His96,His98,Hisl13,Hist63)andZI'(Hisr13,Hist2l,Hist24,Asp 127) are found in CuZn aDd ECSOD acitve site.Cys 107 and Cys 189 forming intrasubunit disulfide bridge found in both protens.Ary 186 found in both.Hjalmarsson, et al. Proc. Na . Acad. Sfl. USA, p. 6343 1987.Carboxy-terminal end of ECSOD is very hydrophilic and contains {+) charged a.a.Marklund proposed this part binds heoarin.SDS gels - MW= 32,000 Da and 29,500 DaBinds to ConA, Ientil, wheat germ lectins. Shows are glycoproteins.A.tloddut ElzyDo ud F6.$o!G.R. Bucttnct PaEe 5GR Bu€ttner tu)Uo)ddant EnzFnes 6IBiologicalMajor SOD in extmcellular fluids such as plasma, lymph, end synovial fluid. Alsofound in tissues. Binds to endothelial cells in vasculature. Involved in inflarnmation.Heparin suppresses inflammation by releasing ECSOD!f. Fe/MnSODFe/MnSOD generally dimersTetramerc found in:Most MnSOD from eukaryotesMnSOD from some bacteriaFeSOD from one bacteriaTrimer MnSOD found in one bacte a.E. coli FeSOD (MW 21,111 Da) 192 a.a.;Human MiSOD (MW - 22,200 Da/monomer) , monomer 196-198 a.a. - a tetramerMost Mn/FeSOD are acidic proteins with pl 4-5Stabilityln general, not as stable as CuZnSODs. Subject to frcczc thaw inactivation IAs pH increases, SOD activity goes down; greater than pH 7.8 activitiy decreasesMn/FeSOD only slowly inactivated by CN; FeSOD inactivated by H2O2.Metal reconstitution studies:Many divalent metals bind to the active sites. Only Mn giv€s activity to MnSOD andonly Fc to FeSOD and not vice versa. Resting metal states present as Mn3+ and Fe3*.g. Svnthesis ofMnSOD in eukawotic cellsHuman liver (Wisp€, BBA 994:30036, 1989.)1. MnSOD is encoded by nuclear chromatin;2. mRNA migmtes to cytosol3. Prot€in is made in ribosomerrntto:t.Llt E!rtE.. dd FbcdonG.R. Bucttncr PaEe 6GRBuettnerturtioxidant Enz],rnesIIIIIII|'tIITMade as a precursor form ririth a MW = 26,000 DaThe precursor is imported post-translationally into mitochondrial matrix. Precursoris clipped by protease in inner mitochondrial membrane to 24,000 Da protein.Proteolytic prccessing is accompanied by energ/ dependent import through themembrane,* Anything blocking ATP formation blocks MnSOD uptake* Blocked by CCCP and NaCNU. Cetelasc (CAT)HistoryThernard. dlscovere! of H2O2, flrst noted tn l8l8 that antmal tissues could decomposeHzoz.l,oew ln l90l introduced the name catalase for the natural compound that decomposesH20z.Wolft and de Stoecklln achteved flrst
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