Investigation of the GTP-binding consensus sequences in Escherichia coli adenylosuccinate synthetase and the enzyme's reaction mechanism

dc.contributor.advisor Herbert J. Fromm
dc.contributor.author Kang, Chulhun
dc.contributor.department Biochemistry, Biophysics and Molecular Biology
dc.date 2018-08-23T07:37:44.000
dc.date.accessioned 2020-06-30T07:08:57Z
dc.date.available 2020-06-30T07:08:57Z
dc.date.copyright Sun Jan 01 00:00:00 UTC 1995
dc.date.issued 1995
dc.description.abstract <p>In this dissertation, on the basis of the similarities in sequence and structure between GTP-binding proteins and AMPSase, it was of interest to determine whether or not the GTP-binding consensus sequences have similar functions. It will be shown that the GTP-binding consensus sequences found in AMPSase play similar roles to those of the GTPase superfamily. The kinetic results show that Lys331 interacts with GTP through hydrophobic interactions between its linear side chain and the aromatic ring of the guanine base of GTP. Also, structural characterization of the G15V mutant using circular dichroism (CD) spectrometry, NMR spectroscopy, and spectrofluorometry indicated that the phosphate-binding region of adenylosuccinate synthetase is involved in a conformational change induced by GTP and IMP binding, and that GTP and IMP binding depend on the pre-existence of other substrates at the active site of the enzyme. The aspartate residue at position 333 of E. coli AMPSase is identified as a key determinant in the recognition of nucleoside triphosphates, and by modest changes in the side chain of the residue 333, the wild-type, GTP-hydrolyzing enzyme is transformed into an even more proficient XTP-hydrolyzing enzyme. A stoichiometric study of the metal ions in AMPSase is presented using kinetics to reveal, for the first time, that the second metal ion is involved in the reaction mechanism of AMPSase through complex formation with aspartate.</p>
dc.format.mimetype application/pdf
dc.identifier archive/lib.dr.iastate.edu/rtd/10949/
dc.identifier.articleid 11948
dc.identifier.contextkey 6430498
dc.identifier.doi https://doi.org/10.31274/rtd-180813-10107
dc.identifier.s3bucket isulib-bepress-aws-west
dc.identifier.submissionpath rtd/10949
dc.identifier.uri https://dr.lib.iastate.edu/handle/20.500.12876/64151
dc.language.iso en
dc.source.bitstream archive/lib.dr.iastate.edu/rtd/10949/r_9540909.pdf|||Fri Jan 14 18:31:33 UTC 2022
dc.subject.disciplines Biochemistry
dc.subject.disciplines Microbiology
dc.subject.disciplines Molecular Biology
dc.subject.keywords Biochemistry and biophysics
dc.subject.keywords Biochemistry
dc.title Investigation of the GTP-binding consensus sequences in Escherichia coli adenylosuccinate synthetase and the enzyme's reaction mechanism
dc.type article
dc.type.genre dissertation
dspace.entity.type Publication
relation.isOrgUnitOfPublication faf0a6cb-16ca-421c-8f48-9fbbd7bc3747
thesis.degree.level dissertation
thesis.degree.name Doctor of Philosophy
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