Transition state and mechanistic study of Trypanosoma cruzi trans-sialidase

190 Pages·2001·6.5 MB·English

by  Yang, Jingsong

TRANSITIONSTATEANDMECHANISTICSTUDYOFTRYPANOSOMACRUZI
TRANS-SIALIDASE
By
JINGSONGYANG
ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL
OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT
OFTHEREQUIREMENTSFORTHEDEGREEOF
DOCTOROFPHILOSOPHY
UNIVERSITYOFFLORIDA
2001
ACKNOWLEDGMENTS
Iamdeeplyindebtedtomyresearchadvisor,Dr.BenjaminA.Horenstein.
Iwouldliketoexpressmysincerestgratitudeforhisguidanceandsupportduring
thecourseofthisprojectandforbeingsuchapatientandhelpingperson. My
appreciation also goes to Dr. Nigel Richards, Dr. Jon Stewart, Dr. David
Silvermanand Dr.WeihongTanforservingontheadvisorycommitteeandfor
giving their time and experience to improve my professional development in
chemistryandbiology.
SpecialthanksgotoDr.SergioSchenkmanforhisgenerosityinproviding
us the plasmids for trans-sialidase overexpression, which started the entire
project.
I wish to express mythanks to the past and present Horenstein group
members-Mike, Eve, Kim, John, Hongbin, Mirela, Katie, Hongyi, and Erin-for
theircompanyandsupport. SpecialthanksgotoMikeandEvefortheirhelpin
the laboratory. I would also liketothank Romaineforprintingthedissertation
andSimonforcomputerhelp. Anappreciationextendstoallmycolleaguesin
theBiochemistryDivision,too.
IamthankfulformyfriendsBaocaiandWentaofortheirhospitalityandfor
allthefuntimewehavebeenenjoyingtogether. Aspecialappreciationisalso
extendedtoallofmyfriendsinUFwhomademystayinGainesvilleapleasant
experience.
I am deeply indebtedto mywife, Nianying, for herpatience, friendship,
supportandloveatalltimes. Withouthersupportnoneofmyaccomplishments
would have been possible. I would also like to thank my daughter, Xinyue
(Sherry),forallthefunandjoywehavebeensharingtogether. Mygratitudealso
goestoallofmyfamilymembersfortheirconstantunderstandingandsupport.
Finally, IwouldliketothanktheNationalScienceFoundationforfunding
and the University of Florida for providing the facilities and an excellent
environmenttocompletethiswork.
in
TABLEOFCONTENTS
page
ACKNOWLEDGMENTS
ii
ABSTRACT vii
CHAPTERS
1. INTRODUCTION 1
Sialicacids 2
Chagas'disease, TrypanosomacruziandTrans-sialidase 8
Glycosylhydrolasesandglycosyltransferases 14
Enzymesthathydrolyseortransfernon-sialosugars:lysozyme
and(3-galactosidases 15
Enzymesthathydrolyseortransfersialosugars:sialidasesand
sialyltransferases 22
MechanisticbackgroundofTrypanosomacruzitrans-sialidase 34
2. RECOMBINANTTRANS-SIALIDASEOVEREXPRESSIONAND
SUBSTRATESYNTHESIS 37
Introduction 37
Results 38
Discussion 43
Overexpressionandpurificationofrecombinanttrans-sialidase 43
Synthesisof([6-3H]Glc)Lactose 46
Synthesisofsialyl-lactoseisotopomers 47
Synthesisofsialyl-galactoseisotopomers 53
Characterizationofsialyl-lactoseandsialyl-galactose
isotopomers 54
Purificationofa-2,3-sialyl-lactosefrombovinecolostrum 55
Synthesisof[3,3'-dideuterio,3H-N-acetyl]sialyl-cc-D-octyl-
galactose 58
Syntheticrouteforthepreparationof([3-3H,3-180)]Gal)sialyl-
galactose 61
Experimental 81
IV
3. KINETICISOTOPEEFFECTSTUDIESONTRANS-SIALIDASE 81
Introduction 81
Kineticisotopeeffectbackground 82
Isotopeeffecttheory 82
Primaryisotopeeffect 89
Secondaryisotopeeffect 90
KIEinenzymaticreactions:commitmenttocatalysis(Cf) 94
KIEmeasurement 95
Thecompetitivemethod 96
Thenon-competitivemethod 99
KIEmethodologyfortrans-sialidase 99
Results 100
Discussion 106
KIEmethodology 106
Kineticparametersforsialyl-galactose 108
Kineticisotopeeffectstudies 108
Conclusions 120
Experimental 121
4. MECHANISTICSTUDIESONTRANS-SIALIDASE 127
Introduction 127
Chemicaltrappingexperiment 127
Initialvelocitystudies 128
Site-directedmutagenesisandchemicalrescuestudies 132
Inhibitiontestsofsialidasetransitionstateanalogsontrans-
sialidase 133
Results 135
Discussion 143
Chemicaltrappingexperiments 143
Initialvelocitystudies 147
Site-directedmutagenesisandchemicalrescuestudies 151
Inhibitiontestsofsialidasetransitionstateanalogsontrans-
sialidase 154
Conclusions 154
Experimental 155
5. CONCLUSIONSANDFUTUREWORK 164
Conclusions ..164
Futurework 155
APPENDICES
167
A 1HNMROFSIALYL-LACTOSESYNTHESIZEDENZYMATICALLY 167
B 1HNMROFSIALYL-GALACTOSESYNTHESIZEDENZYMATICALLY....168
C 1HNMROF4,6-BENZYLIDENE-A-D-METHYLGALACTOSIDE 169
D 1HNMROF2-BENZOYL-4.6-BENZYLIDENE-A-D-METHYL
GALACTOSIDE 170
E 1HNMROF2-BENZOYL-3-KETP-4,6-BENZYLIDENE-A-D-METHYL
GALACTOSIDE 171
REFERENCES 172
BIOGRAPHICALSKETCH 183
VI
AbstractofDissertationPresentedtotheGraduateSchool
oftheUniversityofFloridainPartialFulfillmentofthe
RequirementsfortheDegreeofDoctorofPhilosophy
TRANSITIONSTATEANDMECHANISTICSTUDYOF
TRYPANOSOMACfll/Z/TRANS-SIALIDASE
By
JingsongYang
May,2001
Chair: Dr.BenjaminA.Horenstein
MajorDepartment: Chemistry
Trypanosomacruzitrans-sialidasetransfersthesialicacidgroupfromhost
cellsurfaceglycoconjugatesto parasitesurfaceglycoconjugates ortowater, a
function believed to be involved in the pathogenic process of T. cruzi, the
causative agent of Chagas' disease. Trans-sialidase belongs to a family of
glycosyltransferases whose mechanisms of action have not been well
characterized. This dissertation describes the transition state analysis and
mechanistic study on trans-sialidase with the long term goal of providing
mechanistic informationforthedesign ofspecifictrans-sialidase inhibitors that
mayhaveclinicalapplication.
Thefirstpartofthisworkdescribestheoverexpressionandpurificationof
recombinant trans-sialidase, and the synthesis of a series of substrates
necessaryforthekineticexperiments. Twosubstrates,sialyl-lactoseandsialyl-
vii
galactose,with awidevarietyofisotopiclabels inspecificpositionshavebeen
synthesized. Thesyntheticapproach,purity,yieldandcharacterizationofthese
moleculesispresented.
Thekineticisotopeeffectstudieswiththeabovementionedsubstratesare
discussednext. Theseincludethemeasurementsof13Cprimaryisotopeeffects
and(3-dideuteriosecondaryisotopeeffects. Bothnon-enzymaticsolvolysisand
enzymatictransferreactions have been investigated. The solvolysis reactions
serve as a point of comparison forthe enzyme catalyzed reactions. Kinetic
isotope effects have been measured with both the natural substrate, sialyl-
lactose, and the slow substrate, sialyl-galactose. The results from these
experimentsarecomparedandthetransitionstatestructurefortrans-sialidaseis
proposed.
The dissertation concludes with the discussion of a series of kinetic
experimentsontrans-sialidase. Theseincludeinitialvelocitykinetics,achemical
trapping experiment, site-directed mutagenesis experiments and inhibition
studies. The results of these experiments are discussed and a reaction
mechanismfortrans-sialidaseisproposed.
VIII
CHAPTER
1
INTRODUCTION
TrypanosomacruziisthecausativeagentofhumanChagas'disease,an
epidemicillnessprevalentinCentralandSouthAmerica. T.cruziexpresseson
itssurfacethetrans-sialidaseactivitythattransfersthesialicacidgroupfromhost
cell surface glycoconjugates to its own surface glycoconjugates (transferase
activity)or,lessefficiently,towater(hydrolaseactivity). Itistheonlyenzymethat
creatsglycosidicbondstosialicacidwithoutusingcytidine-5'-monophosphate-N-
acetyl-neuraminic acid (CMP-NeuAc) as the donor substrate. A better
understandingofthemechanismandfunctionoftrans-sialidase maycontribute
tothecontrolofChagas'diseaseandalsomayallowustohaveinsightintowhy
thishomologofthesialidasespreferstransferaseactivityratherthan hydrolase
activity. The work presented in this dissertation represents a study of the
transition state structure and mechanism oftrans-sialidase, with the longterm
goalofprovidingmechanisticinformationforthedesignofspecifictrans-sialidase
inhibitorswithpossibleextensiontotheneuraminidasesandsialidaseswhichare
structurallyhomologous.
2
SialicAcids
Trans-sialidase is involved in the transfer of sialic acid between
glycoconjugates. Itfunctionsbyalteringthedistributionsofsialicacidsonboth
thehostcellandtheparasite'sowncellsurface. Therefore,thebiologicalroles
of trans-sialidase are closely related to the functions of sialic acids. A brief
reviewofthebiologicalfunctionsofsialicacidsisgivenbelowforthepurposeof
helpingreadersbetterunderstandhowtrans-sialidaseactivitymightbeinvolved
inthepathogenicprocessofTrypanosomacruzi.
Sialic acids (figure 1-1) are composed of a family of derivatives of
neuraminic acid (5-amino-3,5-dideoxy-D-g/ycero-D-ga/acto-nonulosonic acid).
Up to now, 36 different sialic acid molecules have been found in various
organisms. Theyare usuallylinkedtothecarbohydratechain ofglycoproteins
and glycolipids via oc-glycosidic bonds (1,2). Sialic acids and sialic acid
conjugatesexhibitavarietyofstructuralfeatures. Sialicacidscanbe linkedto
thepolysaccharidechainviaeithera-2,3,a-2,6,ora-2,8glycosidicbonds(1,2).
Terminalsialicacidsusuallyforma-glycosidicbondsbetweenC-2hydroxylofthe
sialicacidmoleculeandC-3,-4and-6ofthepenultimatenon-sialicacidmoiety,
such as galactose (Gal), N-acetylglucosamine (GlcNAc) and N-
acetylgalactosamine (GalNAc), with the most common linkages being a-2,3to
Galanda-2,6toGalandGalNAc. ThesecanbefoundinbothN-andO-linked
glycoproteins. Sialic acids also attachtoothersialicacid molecules via a-2,8
linkage in oligosialylglycoconjugate and sialylpolysaccharide structures. These
structures are found in bacterial saccharides and glycoproteins as well as in