Smilda, Tiddens

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Proceedings - University of Groningen

   University of GroningenVariation in alcohol dehydrogenase of DrosophilaSmilda, TiddensIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1997Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Smilda, T. (1997). Variation in alcohol dehydrogenase of Drosophila. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 03-06-2022Summary & SamenvattingSummaryThe enzyme alcohol dehydrogenase(ADH) from Drosophila has beenstudied, for many years, by evolutionarybiologists and population geneticists.These studies were mainly focused onwhy and how polymorphism of genesoccurs in populations. The genetic vari-ation in natural populations was primarilyinvestigated by native polyacrylamide gelelectrophoresis, which detects chargedifferences of amino acid sequences ofproteins. Alcohol dehydrogenase of nat-urally occurring Drosophila melanogastervariants and Drosophila simulans ADHdiffer only by a few amino acids. D.melanogaster species can be found inhabitats where alcohol concentrations arerelatively high, whereas D. simulans doesnot tolerate such environments. D. melan-ogaster ADH-S and ADH-F, two wide-spread cosmopolitans, differ only atamino acid position 192. A thermostablevariant D. melanogaster ADH-7Ik has anadditional amino acid substitution atposition 214 (proline to serine). D. simu-lans ADH differs from D. melanogasterADH-S only at positions 1 and 82. Thesesingle or double amino acid substitutionsare responsible for differences in bio-chemical properties. In this thesis thevariation in alcohol dehydrogenase ofthese two sibling species is described.Although no three-dimensional structureof alcohol dehydrogenase from Drosoph-i/a is known, the knowledge of kinetics1 ",')and the structure-function relationshipsshould contribute to the characterizationof this euzyme. Chapter 1 presents ageneral review of the literature of Droso-phila alcohol dehydrogenase.Drosophila ADH belongs to the short-chain dehydrogenases/reductases (SDR), agroup of enzymes with quite differentsubstrate specificities. The three-dimen-sional structures of six short-chaindehydrogenases/reductases have beenelucidated recently and it was shown that,despite a low sequence homology, simi-larity between the three-dimensionalstructures was striking. In Chapter 2 weperformed aligrunent and modellingstudies with Drosophila ADH (DAD}{)and four homologous enzymes belongingto the SDR family with known X-raystructures. The coenzyme binding regionis located in the N-terminal part of theenzymes, whereas the substrate bindingregion is located in the C-terminal one. Aconserved tyrosine and lysine residuetogether with an almost conserved serine139, play an important role in catalysis.This Ser-Tyr-Lys triad is found in almostall short-chain dehydrogenases/reductasesand suggests that these enzymes share acommon catalytic mechanism. There islittle sequence homology in the substratebinding region of short-chain dehydro-genases/reductases, but a substrate-bind-ing loop, present in most enzymes, isresponsible for conformational changesupon substrate binding. This variableSummary & Samenvattiniloop region comprises one or two shortcr-helices and is located in an otherwisequite well conserved part of the structure.Model building of the DADH sequencein this substrate-binding loop suggeststhat in DADH these two helices may alsobe present. The presence of position 192,wherc Drosophila melanogasler ADH-Sand ADH-F differ in sequence, at a cen-tral position in this loop is an intriguingfeature.Chapter 3 describes the purificationmethods used for Drosophila melanogas-ter and Drosophila simulans alcoholdehydrogenase. In the purificationmethod for these enzymes affinity chro-matography with the ligand CibacronBlue is used. Introduction of a secondCibacron Blue affinity chromatographystep with gradient elution with NAD'wasnecessary to obtain pure enzymes. D.melanogaster ADH-S was purified in aslightly different way, because it couldnot be removed from the affinity columnusing high concentrations NAD-. Insteada wash step with a high concentrationNAD' followed by a pH-gradient wasused to obtain the enzyme. It has beenreported in the literature that in homo-geneous enzyme preparations, proteolyticdegradation products appear within ashort period of t ime. However. in ourenzyme preparations no proteolytic degra-dation was found. D. melanogaster ADH-71k and ADH-S have a higher thermo-stability than ADH-F and D. simulansADH.Inhibition experiments with free ligandCibacron Blue showed that D. melan-ogaster ADH-F and ADH-71k have iden-tical inhibition constants (K,) at pH 8.6which are two times higher at pH 9.5.The K, values for D. simulans ADH arethree times lower at both pH values. D.melanogaster ADH-S also showed a lowK, value at pH 8.6, but a relatively highone at pH 9.5. Conditions required forelution of Drosophila alcohol dehydro-genases from the affinity column are inreasonable agreement with K, valuesdetermined for Cibacron Blue and can beexplained from amino acid replacementsbetween the variants. The pH dependenceof the properties of D. melanogasterADH-S can be explained by interactionof a positive charged lysine at position192 with NAD', after closure of asubstrate-binding loop. The Michaelis-Menten constants (K.) for ethanol andNAD* were determined for D. melsn-ogaster alleloenzymes and D. simulansalcohol dehydrogenase and showed to bealmost identical for ethanol, but the KIADvalues differ.In Chapter 4 we show that it is possibleto isolate ADH-FS from heterozygoteDrosophila melanogaster FS flies byaffinity chromatography on CibacronBlue, using first a high concentrationNAD', immediately followed by a pH-gradient. This isolation had to be per-formed at room temperature, becauseNhen it was performed at 4 oC it was notrcssible to separate ADH-FS from ADH-iS and ADH-FF. The inhibition constantf D. melanogasler ADH-FS for Ciba-;ron Blue, at pH 8.6 is very similar to:hat of ADH-SS. However, at pH 9.5 theK, value is only slightly higher, and nowmuch lower than those of ADH-FF andADH-SS. Although there is nocooperativity in inhibitor binding, the twosubunits in D. melanogaster alcoholdehydrogenase influence each other.Negative cooperativity is observed inkinetic experiments on D. melanogasterADH-FS. This can be explained by thepresence of binding sites with differentsubstrate affinities so that the strongerbinding site is occupied first.Dissociation in vitro of D. melanogasterADH-FF and ADH-SS homodimersoccurs during incubation in the presenceof 2 to 3 M urea for 30 minutes at roomtemperature. After removal of urea it ispossible to obtain stable ADH-FS hybridsby reassociation of the monomers.In Chapter 5 the substrate- and stereo-specifities for primary and secondaryalcohols for Drosophila melanogasterADH-F and Drosophila simulqns alcoholdehydrogenase are described. Thesubstrate specificity for D. simulans ADHshowed that the highest activity wasobtained with 1-pentanol (primary alco-hols) and 2-hexanol (secondary alcohols).However, the activity for primary andsecondary alcohols is lower than com-124 -pared to other Drosophila species.Drosophila alcohol dehydrogenase oper-ates by an ordered ternary-complexmechanism with coenzyme (NAD-) as theleading substrate. The rate limiting stepfor secondary alcohols in this mechanismis dissociation of NADH from the finalenzyme-NADH complex. In the presenceof a high concentration NAD' and highconcentrations secondary alcoholssubstrate activation is observed for D.simulans ADH. When substrate activationoccurs for D. simulares ADH, dissociationof NADH from the ternary enzyme-NADH-sec.alcohol complex becomes therate limiting step. The K* values forsubstrate activation are higher, indicatinga lower affinity for secondary alcohols ofthe D. simulans ADH-NADH complexthan of the D. simulqns ADH-NAD-complex.Stereospecificity experiments with R(-)and S(+) enantiomeric forms of second-ary alcohols show that Drosophila simu-lans alcohol dehydrogenase, like otherDrosophila alcohol dehydrogenases pre-fers the R(-) enantiomeric forms of sec-ondary alcohols. From these studies it canbe concluded that the alcohol bindingregion has room for a small and a largehydrophobic side chain of the substrate.Drosophila alcohol dehydrogenase can beconverted in vitro by incubation withNAD- and ketone to a more negativelycharged isoform (Chapter 6). Althoughbeins inactive. the isoforms showedSummary & Samenvatti)activity after native polyacrylamide gelelectrophoresis, but this depends on theketone used. Responsible for the morenegatively charged isoforms is the forma-tion of a covalently bound adductbetween NAD' and ketone. In chapter 6the isolations of ADH isoforms and ofthe negatively charged adducts obtainedin the absence of enzyme by ion-exchange FPLC on a MONO-Q columnare described. When ADH is incubatedwith NAD- and acetylacetone, isoformsappear which have a larger mobility thanthe modified isoforms observed withother ketones. Thermal denaturation andproteolytic degradation of modified ADHisoforms resulted in release of the adduct.Thermal denaturation experiments alsoshowed that modified ADH isoforms aremore stable than the unmodified form asa result of closure of this loop uponsubstrate binding.

Variation in alcohol dehydrogenase of Drosophila

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Variation in alcohol dehydrogenase of Drosophila

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