Distance changes at the regulatory and catalytic sites on Escherichia coli glutamine synthetase: a spin label study on the effect of substrate(s) binding.
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.
Ubom
G A
GA
School of Biochemistry, University of Birmingham, Edgbaston, U.K.
Rhee
S G
SG
Hunt
J B
JB
Chock
P B
PB
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D001665:Binding Sites; D003497:Cyclic N-Oxides / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013113:Spin Labels
D003497:Cyclic N-Oxides; D013113:Spin Labels; C060597:6-(2,2,6,6-tetramethylpiperidine-1-oxyl)-adenosine triphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D005974:Glutamate-Ammonia Ligase