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Studies on Lipoamide Dehydrogenase

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Studies on Lipoamide Dehydrogenase Studieso n Lipoamide Dehydrogenase CENTRALE LANDBOUWCATALOGUS 0000 0489 4321 BffinOTHEtffc t^BOUWUNIVERSEEStt WAGENlNGEfil Promotor : Dr.C .Veege r hoogleraar in de Biochemie Co-promotor : Dr.A .d e Kok universitair hoofddocent JacqiiesA .E .Bene n Studies on Lipoamide Dehydrogenase Proef^chrift ter verkrijging van de graad van doctor in de landbouw- en milieuwetenschappen op geaagva n rector magnificus, Dr. H.C .va nde r Plas, in het ppenbaar te verdedigen op vrijdlag 8 mei 1992 des narniddagst e 13.30 uur in de Aula van daj Landbouwuniversiteit te Wageningen. Aan Anja Aan mijn moeder Ter nagedachtenis van mijn vader fj^optf^ ;v^ Hetverdien t aanbeveling deter m conservatief in relatie tot aminozuursubstituties met meeijomzichtighei d te benaderen. Dit proefschrift, hoofdstuk 2-4. De ejenvoudige manier om redoxpotentialen van flavoproteinen te bepalen zoals voorjesteld door Massey, werkt alleen voor eenvoudige flavoproteinen. Massey, V. (1990) in Flavins and Flavoproteins 1990 (Curti,B. , Ronchi,S .an d Zanetti, G. eds) pp55-66 ,Walte rd e Gruyteran dCo ,Berli n - NewYork . De uit de enzymologie bekende 'negatieve cooperativiteit' blijkt ook bij de Europese unififatie opgeld te doen. Ricard,J .an dCornish-Bowden ,A . (1987) Eur.J. Biochem. 166, 255-272. Devjoorspellend e waarde van de circulair dichroisme methode voor eiwitstructuren is vooijalsnog niet hoog. Provencher, S.W . andGlockner , J. (1981) Biochemistry20, 33-37 . j Wishart, D.S. ,Sykes ,B . D.an d Richards, F. M. (1991) FEBSLet. 293,72-80 . Intggenstellin g tot de bewering van Scrutton etal. isd e afwezigheid van reductie van het flavine in Cys47->Ser gemuteerd E. coliglutathio n reductase door NADPH niette verWachten. Scrutton, N.S. , Berry ,A. , Deonarian,M . P.an dPerha m ,R . N.(1990 ) Proc. R. Soc. London242, 217-224. De ^ewering door Borges ef al. dat in Pseudomonasfluorescens ee n tweede gen voor lipoimide dehydrogenase zou zijn aangetoond door Benen etal. i s tout. Borges,A. , Hawkins,C . F., Packman,L C.an d Perham,R . N. (1990) Eur.J. Biochem. 194, 95-102. Benen,J .A . E.,Va n Berkel,W .J . H.,Va n Dongen , W. M.A . M., Muller, F.an dD e Kok ,A . (1989) J. GenMicrobiol. 135, 1787-1797. 4<D<p TY De opvatting dat alle flavine-afhankelijke oxidases een flavine-N5-sulfiet adduct vormen verdient aanpassing. Muller,F . (1987) Free RadicalBiology Medicine 3, 215-230 . De verwachte 5%dalin g van verkeersslachtoffers, ten gevolge van de maatregel overdag verplicht licht te voeren op alie gemotoriseerde vervoermiddelen, is niet gecorrigeerdvoo r de te verwachtentoenam e van het aantal slachtoffers onder motorrijders. De excessieve veiligheidsmaatregelen waarmee asbest is omgeven zal, gezien de maatregelente n aanzien van AIDS preventie, ertoe leiden dat het afsluiten van een levensverzekering gepaard zai gaan met een asbest-antecedenten onderzoek. Stellingen behorende bij het proefschrift 'Studies on lipoamidedehydrogenase ' Wageningen, 8 mei 1992. Jacques A. E.Benen . Voonvoord Graagwi l ik op deze plaats iedereen bedanken die bij de totstandkoming van dit proefs^hrift heeft bijgedragen. Ce^s Veeger, mijn promotor, met wie pittige discussies werden gevoerd over ook 'zijn' efizym. Aart de Kok die alsco-promoto r optrad.Aar t bedankt voor je geduldig oor envoo r de vrijleid die je me hebt gelaten in onderzoek en vorming. WiHem van Berkel bij wie menig wetenschappelijk proeballonnetje werd opgelaten dat va|tk werd doorgeprikt endi e veel heeft bijgedragen aan het hele proefschrift. Adlie Westphal, die het gen coderend voor lipoamide dehydrogenase cloneerde, niet alleen een fijn collega en kamergenoot maar ook een gezellige kerel buiten werktijd. Miriam Gilissen, Fenneke Linker, Erna Hissink, Nicole Dieteren en Sandra Japenga die in het kader van een afstudeervak aan verschillende aspecten van lipoamide dehydrogenase werkten. Zdzislaw Zak who spent three months ontediou stitration s of mutated enzymes and who racordedth e CDspectra . Charles Williams Jr. who invited met o visit his lab inAn n Arbor, Michigan, USA,i n order to be able to do rapid scan analysis. DavidArscot t who made me familiar with the cofriplex apparatus. Charles and Davidthank s a lot. Without this oppertunity, fruitful{cooperatio n anddiscussion s Chapter 3woul d have had less impact. Ihop e the cooperation will be continued inth e future. Walter van Dongen die de correctie van hoofdstuk 5voo r zijn rekening nam. Andrea Mattevi, Bram Schierbeek en Wim Hoiva n de kristallografiegroep van de univer$iteit van Groningen die de structuur beschikbaar stelden voordat deze gepubliceerd was en die aanzienlijk meer mutaties voorstelden dan een promovendus kan maken en bestuderen. Allf medewerkers van de afdeling Biochemiedi e ervoor zorgden dat er altijd een prima|werksfeertj e was en waarbijj e je gal kon spuwen als het weer eens mis ging. Mijfi moeder,famili e en vrienden die altijd gei'nteresseerdware n in de voortgang. Ante,di e nooit klaagde als ikwee r te laat thuis was of in het weekend moest werken. Die me intijde n vantegensla g atijd uit de put hielp, kortom,di e een gewelaige steun voor mewas .Anj a bedankt I Chapters 2 and 5 have been published andchapter s 3 and 4 will be published separately. Chapter 2: Benen,J. , Van Berkel,W. ,Zak ,Z. ,Visser ,T. ,Veeger ,C . andd e Kok,A . (1991) Eur.J. Biochem. 202,863-872 . Chapter 3: Benen,J. , van Berkel,W. ,Dieteren , N.,Arscott , D.,William sJr. , C, Veeger, C. andd e Kok,A . (1992) Eur.J. Biochem.,(submitted ) Chapter 4: Benen,J. , Van Berkel,W. ,Veeger , Can dd e Kok,A . (1992) Eur. J. Biochem.,(submitted ) Chapter 5: Benen, J.A . E.,Va n Berkel,W .J .H. ,Va n Dongen,W . M.A . M.,Muller ,F . andd e Kok,A . (1989) J. Gen.Microbiol. 135, 1787-1797. The research described inthi s thesis was carried out atth e Department of Biochemistry, Agricultural University, Wageningen,Th e Netherlands, under the direction of Prof. Dr. C. Veeger. The investigations were financially supported by the Netherlands Organisation for Scientific Research (N.W . O.) underth e auspices of the Foundation for Chemical Research (S.O.N.). Contents Page Abbreviations 9 List ofjenzyme s 10 Chapter 1. Introduction 11 Chaptfr 2. Lipoamide dehydrogenase from Azotobacter vinelandii: 43 site directed mutagenesis of the His450-Glu455 diad. Spectral properties of wild-type and mutated enzymes. Chapter 3. Lipoamide dehydrogenase from Azotobacter vinelandii: 53 site directed mutagenesis of the His450-Glu455 diad. Kinetics of wild-type and mutated enzymes. Chapter 4. Lipoamide dehydrogenase from Azotobacter vinelandii: 79 Role of the C-terminus in catalysis and dimer stabilization. Chapter 5. Molecular cloning and sequence determination of the 95 /pd-gene encoding lipoamide dehydrogenase from Pseudomonas fluorescens. Chaptor 6. Lipoamide dehydrogenase from Pseudomonas fluorescens. 107 Acharacterizatio n of the enzyme. General discussion 115 Samenvatting 123 Curriculum vitae 131 List of publications 131 Abbreviationi s AAD+ 3-aminopyridine adenine dinucleotide AcPyA0e+ 3-acetylpyridine adenine dinucleotide AcPyApeH 3-acetylpyridine adenine dinucleotide reduced BCOA0C branched-chain oxo acid dehydrogenase complex Cl2lndi 2,6-dichlorophenolindophenol Cm midpoint concentration of Gdn/HCI required for unfolding CT 530 nmcharg e transfer species. AG standard Gibbs energy Eox oxidized (mutated) lipoamide dehydrogenase EH2 2-electron reduced (mutated) lipoamide dehydrogenase with disulfide reduced (530 nmcharge-transfer - and tautomeric species). EH- EH2on e step deprotonated E2- EH2tw o steps deprotonated EH4 general descriptive term for 4-electron reduced (mutated) lipoamide dehydrogenase EDTA ethylene diamine tetraacetate Em midpoint redox potential FAD(Hf) flavin adenine dinucleotide (reduced) FL fluorescent species. FIH- 2 electron reduced species with electrons on the flavin. Gdn/H0l guanidinium hydrochloride h Hill coefficient Hepps^EDTA Hepps/EDTA buffer, pH 8.0 k rate constant kb kilo basepairs kcat turnover rate,|j.mol e product per active site persecond . Kd dissociation constant K| inhibition constant Km Michaelis Menten constant LipS2 D,L - 6,8 -thiocti c acidamide ; lipoamide Lip(SH)2 reduced lipoamide /pd-gehe gene encoding lipoamide dehydrogenase NAD(R)+ nicotinamide dinucleotide (phosphate) oxidized NAD(Fl)H nicotinamide dinucleotide (phosphate) reduced Meq (sj>. Molar equivalent(s) Mr relative molecular mass Nbs2 5,5'-dithiobis(2-nitrobenzoate) nm nanometer PDC pyruvate dehydrogenase complex pKa -log(proton dissociation constant) PPi/EDTA-buffer 50 mMsodiu m pyrophosphate, 0.5 mMEDTA , pH 8.0 PrtX unknown protein encoded by an open reading frame downstream ofth e /pcfgen e in Azotobacter vinelandiian d Pseudomonas fluorescens OGDC 2-oxo glutarate dehydrogenase complex ORF open reading frame SDS sodiumdodecylsulfate 6xSSC 0.9 MNaCI ,0.0 9 MNa 3citrate-2 H20, pH 7.0 SSE 20 %(w/v ) sucrose, 150 mMNaCI , 100 mMEDTA , pH 7.6 SucA gene encoding 2-oxo glutarate dehydrogenase SucB gene encoding succinyl transferase 4n melting temperature U unit of activity: 1umol e product per minute v apparent velocity, nmoles product per mgenzym e per minute V maximal velocity, u.moles product per mg enzyme per minute List of enzymes 1. lipoamide dehydrogenase, NADH:lipoamide oxidoreductase (EC. 1.8.1.4) 2. DNA restriction endonucleases Alul,SamH1 , EcoR1, Hinc\\,Hind\\\, Kpn\, Notl, Psfl, Sau3A I,Sma\, Sph\, Ssti, (EC 3.1.24.4) 3. DNA polymerase I(Kleno w fragment)(EC 2.7.7.7) 4. DNA ligase (EC6.5.1.1 ) 5. T4-DNA polymerase (EC 2.7.7.7) 6. Glucose-6-phosphate dehydrogenase, D-Glucose-6-phosphate: NADP 1- oxidoreductase (EC 1.1.1.49) 7. Xanthine oxidase, xanthine: oxygen oxidoreductase (EC 1.1.3.22) 10 Chapter 1 Introduction Occurrence and function of lipoamide dehydrogenase Lipoamide dehydrogenase is the common name for NADH:lipoamide oxido- reductase (EC 1.8.1.4).Th e enzyme wasfirs t described by Straub [1] and has been knownjfo ryear s as Straub diaphorase as it readily transfers electrons from NADHt o artificial electron acceptors like 2,6-dichlorophenolindophenol and methylene blue [2].
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