PMID:1672611
| Citation |
Ubom, GA, Rhee, SG, Hunt, JB and Chock, PB (1991) Distance changes at the regulatory and catalytic sites on Escherichia coli glutamine synthetase: a spin label study on the effect of substrate(s) binding. Biochim. Biophys. Acta 1077:91-8 |
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| Abstract |
A spin-labeled ATP analogue, 2,2,6,6-tetramethylpiperidine-1-oxyl adenosine triphosphatase (Tempo-ATP) is used to adenylate Escherichia coli glutamine synthetase (L-glutamine: ammonia ligase (ADP-forming), EC 6.3.1.2). The Tempo adenylylated glutamine synthetase (Tempo-GS) exhibits similar catalytic properties, pH profile and inhibitor susceptibility as those of glutamine synthetase adenylylated with normal ATP. Using the spin label on the enzyme as a probe and employing the spin-spin interactions between the label probe and paramagnetic Mn2+, the distances from the nitroxyl moiety of the covalently bound Tempo-AMP to the two Mn2+ binding sites, n1 and n2 were determined. The n1 site is the structural site and n2 is located at the catalytic site. The distances from Mn2+ at n1 and n2 sites to the nitroxyl radical are 19 and 16 A, respectively. Binding of the substrate, L-Glu, causes a protein conformational change which is reflected by the reduction of approximately 2 A for the n1 to Tempo-AMP distance and lengthening of approximately 2 A for the n2 to the Tempo-AMP distance. Addition of ATP to the Tempo-GS/L-Glu complex increases the distance between n1 and Tempo-AMP, and n2 and Tempo-AMP by 4 and 3 A, respectively. |
| Links | |
| Keywords |
Adenosine Diphosphate/metabolism; Adenosine Triphosphate/analogs & derivatives; Adenosine Triphosphate/metabolism; Binding Sites; Cyclic N-Oxides/metabolism; Electron Spin Resonance Spectroscopy; Escherichia coli/enzymology; Glutamate-Ammonia Ligase/chemistry; Glutamate-Ammonia Ligase/metabolism; Kinetics; Magnetic Resonance Spectroscopy; Protein Conformation; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Spin Labels |
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