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

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The paramagnetic effect of Mn(II) on 13C and 31P nuclear magnetic resonance signals from the [2-13C]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, n1 and n2. Binding of Mn(II) to the n1 site converts an inactive apo-enzyme to its active form, while the metal ion bound at n2 occupies the metal-nucleotide substrate site. The distances from Mn(II) at the n1 and n2 sites to phosphorus are ∼10 and ∼7 Å and to the 2-C position of the adenine ring are ∼12 and ∼11 Å, respectively. The fluorescence energy transfer method was used to determine distances between Co(II) at n1 and n2 and the adenylyl site. For this experiment the enzyme was adenylylated with ε-ATP. The distances between ε-adenine and Co(II) at n1 and n2 are ∼13 and ∼11 Å, respectively. Quantitation of the paramagnetic effect due to Co(II) on the 31P nuclear magnetic resonance signal yielded values of 8 and 6 Å for the distances between the phosphorus of the covalently bound AMP and the n1 and n2 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.

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