Topographical analysis of regulatory and metal ion binding sites on glutamine synthetase from Escherichia coli: 13C and 31P nuclear magnetic resonance and fluorescence energy transfer study.
The paramagnetic effect of Mn(II) on (13)C and (31)P nuclear magnetic resonance signals from the [2-(13)C]ATP adenylylated glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming); EC 6.3.1.2] from Escherichia coli was measured. This effect permitted the determination of distances from the 2-C position and the phosphorus of covalently bound AMP to the two Mn(II) binding sites, n(1) and n(2). Binding of Mn(II) to the n(1) site converts an inactive apo-enzyme to its active form, while the metal ion bound at n(2) occupies the metal-nucleotide substrate site. The distances from Mn(II) at the n(1) and n(2) sites to phosphorus are approximately 10 and approximately 7 A and to the 2-C position of the adenine ring are approximately 12 and approximately 11 A, respectively. The fluorescence energy transfer method was used to determine distances between Co(II) at n(1) and n(2) and the adenylyl site. For this experiment the enzyme was adenylylated with epsilon-ATP. The distances between epsilon-adenine and Co(II) at n(1) and n(2) are approximately 13 and approximately 11 A, respectively. Quantitation of the paramagnetic effect due to Co(II) on the (31)P nuclear magnetic resonance signal yielded values of 8 and 6 A for the distances between the phosphorus of the covalently bound AMP and the n(1) and n(2) sites, respectively. The results reveal that the covalent modification site is very close to the catalytic center of the enzyme. In this study both nuclear magnetic resonance and fluorescence energy transfer techniques have been used to determine distances between the same set of sites on an enzyme surface.
D000249:Adenosine Monophosphate / Q000378:metabolism; D001665:Binding Sites; D003035:Cobalt / Q000378:metabolism; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008345:Manganese / Q000378:metabolism; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D003035:Cobalt; D000249:Adenosine Monophosphate; D008345:Manganese; D005974:Glutamate-Ammonia Ligase