Physical studies of adenylosuccinate synthetase

dc.contributor.author	Bass, Michael
dc.contributor.department	Biochemistry, Biophysics and Molecular Biology
dc.date	2018-08-17T12:02:27.000
dc.date.accessioned	2020-07-02T06:07:23Z
dc.date.available	2020-07-02T06:07:23Z
dc.date.copyright	Thu Jan 01 00:00:00 UTC 1987
dc.date.issued	1987
dc.description.abstract	<p>To determine the chemical mechanism of the reaction catalyzed by adenylosuccinate synthetase, positional isotope exchange studies were performed. Positional isotope exchange from the (beta)-(gamma) bridge to the (beta) nonbridge position of (gamma)-('18)O GTP was followed using ('31)P NMR. The positional isotope exchange was found to occur in the presence of either IMP or IMP and succinate. The exchange did not occur in the presence of asparate. These results support a reaction mechanism which involves formation of a 6-phosphoryl-IMP intermediate with subsequent attack by aspartate to form adenylosuccinate as originally proposed by Lieberman in 1956 Lieberman, I. J. Biol. Chem., 1956, 223, 327-339 ;In order to resolve the NMR resonances for positional isotope exchange, it was necessary to find a chelator which would limit exchange broadening. trans-1,2-Diaminocyclohexane-N,N,N',N'-tetraacetic acid was found to be a suitable chelator at neutral and acidic pH;Studies of adenylosuccinate synthetase from Escherichia coli have been limited by the low concentrations of enzyme present in the cell and the difficulty in purifying the enzyme to homogeneity. Overproduction of the enzyme by cloning the purA gene into a runaway replication plasmid allowed the cells to produce a much higher concentration of enzyme. A new purification scheme is reported that takes advantage of the overproduced enzyme. Yields of 75 mg of homogeneous enzyme have been obtained from 76 g of E. coli cell paste.</p>
dc.format.mimetype	application/pdf
dc.identifier	archive/lib.dr.iastate.edu/rtd/8512/
dc.identifier.articleid	9511
dc.identifier.contextkey	6335266
dc.identifier.doi	https://doi.org/10.31274/rtd-180813-10925
dc.identifier.s3bucket	isulib-bepress-aws-west
dc.identifier.submissionpath	rtd/8512
dc.identifier.uri	https://dr.lib.iastate.edu/handle/20.500.12876/81509
dc.language.iso	en
dc.source.bitstream	archive/lib.dr.iastate.edu/rtd/8512/r_8716742.pdf\|\|\|Sat Jan 15 02:12:56 UTC 2022
dc.subject.disciplines	Biochemistry
dc.subject.keywords	Biochemistry and biophysics
dc.subject.keywords	Biochemistry
dc.title	Physical studies of adenylosuccinate synthetase
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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