Physical studies of adenylosuccinate synthetase Bass, Michael
dc.contributor.department Biochemistry, Biophysics and Molecular Biology 2018-08-17T12:02:27.000 2020-07-02T06:07:23Z 2020-07-02T06:07:23Z Thu Jan 01 00:00:00 UTC 1987 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>
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dc.identifier.articleid 9511
dc.identifier.contextkey 6335266
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dc.language.iso en
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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 dissertation Doctor of Philosophy
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