EPR Investigation of the Mn(II) Binding Sites in Glutamine Synthetase (Escherichia coli W)

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Abstract
The nature of the intermediate-affinity (n2) Mn(II) binding sites in glutamine synthetase [EC 6.3.1.2] has been studied as a function of adenylylation in a variety of enzyme-metal complexes by EPR. In the absence of nucleotide, the n2 Mn(II) environment is nearly isotropic, the Mn(II) bonds are highly ionic, and the interaction distance R≥12–14 A. Nucleotide binding at the n2 Mn(II) site renders the n2 Mn(II) signal unobservable and causes a reduction in signal amplitude (∼30%) and line broadening (∼6 G) at the high-affinity (n1 Mn(II) site. This behavior indicates that nucleotide binding induces a conformational change in the enzyme which brings the previously distant n1 and n2 sites into closer proximity (R≤8–11 A), possibly for the purpose of activating the nucleotide for direct phosphoryl transfer to L-glutamate. In line with this suggestion, the broad, unresolved resonances in complexes containing both L-methionine SR-sulfoximine (MSOX) and nucleotide may result from the phosphorylation of MSOX. The n2 Mn(II) site is not affected by adenylylation in all the enzyme-metal complexes studied, which suggests that the regulatory effects of adenylylation may only act at the n1 Mn(II) sites.