Degree Type
Dissertation
Date of Award
1987
Degree Name
Doctor of Philosophy
Department
Biochemistry, Biophysics and Molecular Biology
Abstract
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.
DOI
https://doi.org/10.31274/rtd-180813-10925
Publisher
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Copyright Owner
Michael Brian Bass
Copyright Date
1987
Language
en
Proquest ID
AAI8716742
File Format
application/pdf
File Size
96 pages
Recommended Citation
Bass, Michael Brian, "Physical studies of adenylosuccinate synthetase " (1987). Retrospective Theses and Dissertations. 8512.
https://lib.dr.iastate.edu/rtd/8512