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An investigation to explore the role of the C-terminal helical thong in the stability of the Escherichia coli glutamine synthetase dodecamer and to explore the minimum quaternary structure of glutamine synthetase needed for the regulatory adenylylation reaction
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An investigation to explore the role of the C-terminal helical thong in the stability of the Escherichia coli glutamine synthetase dodecamer and to explore the minimum quaternary structure of glutamine synthetase needed for the regulatory adenylylation reaction
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An investigation to explore the role of the C-terminal helical thong in the stability of the Escherichia coli glutamine synthetase dodecamer and to explore the minimum quaternary structure of glutamine synthetase needed for the regulatory adenylylation reaction
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Dissertation
An investigation to explore the role of the C-terminal helical thong in the stability of the Escherichia coli glutamine synthetase dodecamer and to explore the minimum quaternary structure of glutamine synthetase needed for the regulatory adenylylation reaction
1994
Overview
The role of the C-terminal helical thong in the stability of the E-coli glutamine synthetase dodecamer and the minimum glutamine synthetase structure needed for the regulatory adenylylation reaction were investigated. In the first part of this study, three site-directed mutants were created that truncate the helical thong of E-coli glutamine synthetase by 8, 3, or 1 amino acid(s) and were E461Stop, E463Stop, and V468Stop. The V468Stop mutant was dodecameric as seen from non-denaturing gel electrophoresis but the E463Stop and E461Stop mutants were mostly hexameric. There was a considerable amount of dodecamer present for E463Stop enzyme and the dodecamer for this mutant slowly dissociated to the hexameric form. The E461Stop hexamer and the E463Stop hexamer showed very little activity and neither hexamer could be adenylylated by adenylyltransferase (ATase). The E463Stop dodecamer, however, was active and could be adenylylated by ATase. The steady-state kinetic parameters showed that with increasing truncation there was a decrease in the specificity constant and k$\\sb{\\rm cat}$ for the dodecamers. The second part of the study addressed the question of the minimum quaternary structure of GS needed for the regulatory adenylylation reaction. To determine the amount of quaternary structure necessary for adenylylation, wild-type dodecamer (partially inactivated with 12% MSOX) was dissociated into a distribution of dodecamer, decamer, octamer, hexamer, and tetramer (GS Ladder) and used in the adenylylation reaction. Each of the GS ladder oligomers was adenylylated so the dodecameric structure was not essential for adenylylation. The adenylylation reaction was used next to determine the ratio of AMP bound per GS subunit for each of the oligomers. The ratios worked out to be 1.0 for the dodecamer, 0.75 for the octamer, 0.68 for the hexamer, and 0.47 for the tetramer after 6 hours of incubation. These ratios suggest that ATase needs the tetramer face of the dodecamer for adenylylation.
