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Pyruvate Dehydrogenase Pyruvate dehydrogenase Its structure, function andinteraction s withinth e pyruvate dehydrogenase multienzyme complex. Promotoren: ProfDrC. Veeger Hoogleraar ind ebiochemi e Co-promotor: Dr. Ad eKo k Universitair hoofddocent Laboratoriumvoo r Biochemie Promotie commissie Dr.V .Bunnik ,Bir k Medical Research Institute,Ne w York Prof.Dr .W.G.J . Hoi, Washington University, Seattle Prof.Dr .J.F .Koster ,Erasmu s Universiteit, Rotterdam Prof.Dr .A.J.W.G . Visser, Wageningen Universiteit Prof. Dr.W.M . deVos ,Wageninge n Universiteit Prof.Dr .S.C .d eVries , Wageningen Universiteit M^<7(' Pyruvate dehydrogenase Its structure,functio n andinteraction s withinth e pyruvate dehydrogenase multienzyme complex. Annechien Frederique Hengeveld Proefschrift terverkrijgin g van degraa dva n doctor opgeza gva n derecto r magnificus vanWageninge n Universiteit Prof. Dr. Ir.L .Speelma n inhe t openbaart e verdedigen opwoensda g2 4 april 2002 desnamiddag so mhal f twee ind eAul a (-.-y- ISBN: 90-5808-620-8 Stellingen 1. Opbasi sva n dehog ehomologi etusse n prokaryotepyruvaa t dehydrogenases (Elp) uit pyruvaat dehydrogenase multienzymcomple xka ngeextrapoleer d worden dat prokaryoot Elp bindt aand ecentral e component (E2p)vi aee nN-terminaa l bindingsdomein. Dit proefschrift 2. Opbasi sva nd evoorgesteld e wijze vanbindin gvoo rheterotetramee r Elb zijn erzee r grote verschillen tussen dewijz e van binding van El aankubisc h E2(homodimere naa n een 24-meer) enaa n dodecaedrisch E2 (heterotetrameren aan een 60-meer),d e voorgestelde wijze vanbindin g vanheterotetramee r El behoeft echter meer experimentele ondersteuning. Ditproefschrift &Mvarsson, A., Seger,K., Turley,S., Sokatch, JR. and Hoi, W.G.J.: Crystal structure of 2- oxoisovalerate and dehydrogenase and the architecture of2-oxo acid dehydrogenase multienzyme complexes (1999)Nat. Struct.Biol. 6, 785-792 3. Deovergan gva n enzymologienaa rfarmacologi e isvoora l moeilijk omdate ri n verschillende "talen"ove r dezelfde zakenword t gediscussieerd. 4. Eiwithomologi e modelling zonder experimentele validatie isnie t meer dan speculeren over de structuurva n een eiwit. Omdez erede n zou eraltij d eennauw e samenwerking moetenzij n tussen theoretici enexperimentel e biochemici / biofysici. 5. Alhoewel het ophelderen van de structuur vanrhodopsin e een enormebijdrag e heeft geleverd aan inzicht inhe tmechanism e van G-eiwit gekoppelde receptoren moet voorzichtigheid inach tworde n genomenbi j hetextrapolere n vandez e structuur naar andereG-eiwi tgekoppeld e receptoren. Palczewski, K.,Kumasaka, T., Hori, T.,Behnke, C.A., Motoshima, H., Fox, B.A., Le Trong, J.,Teller, D. C, Okada, T.,Stenkamp, R. E., Yamamoto,M., Miyano, M.: Crystal Structure of Rhodopsin: A G Protein- CoupledReceptor (2000)Science 289, 739 6. Hetbete r omi nee n goed milieu te leven dan omui t een goed milieu tekomen . 7. Omdathe tkrijge n van eenkin d altijd slecht uitkomtbinne n een wetenschappelijke carriere, isdi t geen reden omhe tkrijge n van kinderen uitt e stellen. 8. Elkeplan t isee n waterplant. Stellingen behorende bij het proefschrift 'Pyruvate dehydrogenase. Itsstructure , function and interactions within the pyruvate dehydrogenase multienzyme complex.' Annechien F. Hengeveld Wageningen, 24apri l200 2 Contents Abbreviations and symbols 1 General introduction 1 Expression andcharacterisatio n ofth ehomodimeri c E1-componen t of the ._ Azotobactervinelandii pyruvat e dehydrogenase complex. Pyruvate dehydrogenase from Azotobactervinelandii: Properties ofth eN - ,, terminally truncated enzyme. Functional and structural characterisation ofth eN-termina l domain of 4 53 pyruvate dehydrogenase. Identification ofth eE2-bindin g residues inth eN-termina l domain of „q prokaryotic pyruvate dehydrogenase. A structural alignment ofth ehomodimeri c andheterotetrameri c2 - „„ oxoacid dehydrogenases andtransketolase . Structural basis ofth e dysfunctioning ofhuma n 2-oxoaci d dehydrogenase „ complexes. 8 Summary and concluding remarks. 127 References 133 Samenvatting 141 Nawoord 147 Curriculum vitae 148 List ofpublication s 149 Abbreviations andsymbol s AD Alzheimer's disease ADHC Acetoin dehydrogenase complex BCAA Branched-chain amino acid BCDC Branched-chain oxoacid dehydrogenase complex BCKA Branched- chain a-ketoacid CAT Chloramphenicol acetyltransferase Cblnd 2,6-dichlorophenol-indophenol El a-ketoacid dehydrogenase component Elb Branched-chain a-ketoacid dehydrogenase (EC 1.2.1.25) Elo 2-oxoglutaratedehydrogenas e (EC 1.2.4.2) Elp Pyruvate dehydrogenase (EC 1.2.4.1) E2 Dihydrolipoyl acyltransferase component E2p Dihydrolipoyl transacetylase (EC2.3.1.12 ) E3 Dihydrolipoamide dehydrogenase component (EC 1.8.1.4) E3BP E3bindin g protein (protein X) GDHC Glycine dehydrogenase complex LS Leigh's syndrome MP-8 Microperoxidase 8 MSUD Maple Syrup Urine Disease NOE Nuclear Overhauser effect Nterm-Elp Peptide representing amino acid 4t o 45o f Elp from Azotobacter vinelandii OGDC Oxoglutarate dehydrogenase complex PDC Pyruvate decarboxylase PDHC Pyruvate dehydrogenase multienzyme complex PFOR Pyruvate ferredoxin oxidoreductase POX Pyruvate dehydrogenase ThDP Thiamin diphosphate TK transketolase TPPI time proportional phase incrementation 1 Introduction Pyruvate dehydrogenase multi-enzyme complex (PDHC) is member of a family of multi- enzyme complexes that catalyse the irreversible decarboxylation of various 2-oxoacid substrates to their corresponding acyl-CoA derivatives,NAD H and CO2.PDHC , like the other members of this family, consists of an assembly of non-covalently attached proteins that catalyse successive steps in a reaction sequence. The combined catalytic activity of the enzymes in the assembly is greatly enhanced through the coupling of the successive reactions of the complex. Covalent attachment of reaction intermediates forces them to complete the reaction sequence and prevents "scavenging" by competing enzymes. Additionally, active site coupling and substrate channelling limit the diffusion of the substrates and degradation of unstable reaction intermediates. The coupling ofth e successive reaction steps finally results in a very effective regulation of the overall reaction sequence that can occur on all participating enzymes [Reed, 1974;Hammes , 1981]. 2-oxoacid dehydrogenase complexes hold keypoint s in primary energymetabolis m (figure 1): PDHC at the branch-point of glycolysis and citric acid cycle, the oxoglutarate dehydrogenase complex (OGDC) in the citric acid cycle,th e branched-chain oxoacid dehydrogenase complex (BCDC) inamin o acid catabolism,th e glycine dehydrogenase complex (GDHC) inth e glycine cleavage system and in the photosynthetic carbon cycle and acetoin dehydrogenase complex (ADHC) in acetoin metabolism. Malfunctioning of any of these complexes in man leads to a broad variety of clinical manifestations [Wynn et al., 1996;Ind o &Matsuda , 1996;Ker r et al., 1996; Danner & McKean, 1996; Chuang & Shih 1995; Robinson, 1995; Kerr & Zinn 1995; Dahl, 1995; Patel & Harris 1995; Roche & Patel, 1989; Kish, 1997; Mizuno et al., 1994]. Consequently, there is a broad interest in the functioning of these complexes on a molecular level. This thesis describes the studies on the pyruvate dehydrogenase (Elp) component of the PDHC from Azotobacter vinelandii, a gram-negative bacterium. PDHC from Azotobacter vinelandii is of special interest as it is the smallest and simplest know 2-oxoacid dehydrogenase. Consequently, in this introduction I will mainly focus on the structure and Chapter 1 branched chain glucose glycine amino acid * pyruvate GDHC branched chain glycine ct-keto acid V PDHC -* serine-*—-'- N3,N10-methylene BCDC tetrahydrofolate V acetyl-Co A branched chain •4 acyl-CoA "^ acetoacetate- Krebs cycle propionyl-CoA a -ketoglutarate OGDC "* succinyl-CoA' Figure 1. Metabolic location of2-oxoaci d dehydrogenase complexes [Olson, 1989]. function of PDHC from gram-negative bacteria. The functioning and dysfunctioning of human 2-oxoacid dehydrogenase multi-enzyme complexes and their involvement in metabolic disease isreviewe d in chapter 7. Reaction mechanism and architecture Five 2-oxoacid dehydrogenase complexes are found in gram-negative bacteria: PDHC, OGDC, BCDC, GDHC and ADHC. All, except GDHC [Douce etal. , 2001], share acommo n architecture. The complexes consist of multiple copies of three enzymes: 2-oxoacid dehydrogenase or El-component, dihydrolipoyl acyltransferase (E2)an d dihydrolipoamide dehydrogenase (E3). Mammalian and yeast PDHC contain a so-called E3-binding protein (E3BP, formerly called protein X) [de Marcucci & Lindsay, 1985; Lawson et al., 1991; Neagle & Lindsay, 1991] that is involved inth ebindin g of E3 toth ecomplex . Eukaryotic PDHC and BCDC in addition, contain specific regulatory proteins (El-kinase and El phosphatase) [Linn et al., 1969; Patel & Roche, 1990]. E2 forms the central core ofth e complex with either octahedral (24 subunits) or icosahedral (60 subunits) symmetry. Multiple Introduction copies of the peripheral components El and E3 are non-covalently attached to this core. The resulting multi-enzyme complexes are of such an enormous size (-5-14 MDa)tha t complexes Figure 2. Reaction mechanism of 2- oxoacid dehydrogenase complexes. A 2- oxo acid dehydrogenase (Elp, Elb or Elo) uses thiamin diphosphate (ThDP) to carry out the oxidative R-C-COOH decarboxylation of the substrate, forming El an acylated lipoic acid, covalently attached to the acyltransferase component (E2p, E2b or E2o) and C02. PDHC: R=CH3 OGDC: R= HOOC-CH z-CHz The E2-component catalyses the transfer BCDC:
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