Manganese(II) and substrate interaction with unadenylylated glutamine synthetase (Escherichia coli W). II. Electron paramagnetic resonance and nuclear magnetic resonance studies of enzyme-bound manganese(II) with substrates and a potential transition-state analogue, methionine sulfoximine

Abstract

The enhancement of the longitudinal proton relaxation rate of solvent H2O protons which occurs when Mn(II) is bound to the tight metal ion site of unadenylylated glutamine synthetase (GS) was used to determine the binding constant of L-methionine (SR)-sulfoximine to GS-Mn(II) complexes. The binary enhancement for GS-Mn(II) is 22 at 24 MHz, 25.degree. C. The enhancement is lowered in the presence of the sulfoximine and the computed dissociation constant is 30 .mu.M with .epsilon.t, the enhancement for the ternary complex, equal to 3.0. Titration curves for the sulfoximine were also obtained in the presence of Mg-ADP, Mg-ADP plus Pi, and Mg-ATP. The dissociation constants were 9, 5 and 0.8 .mu.M, respectively. The progressive tightening of the dissociation constants is symptomatic of conformational changes at the active site as the total subsite occupied by ATP is filled. The number of rapidly exchanging H2O molecules drops from 2 to .apprx. 0.1 when saturating concentrations of L-methionine (SR)-sulfoximine and nucleotide are present. The kinetically determined KI value of .apprx. 4 .mu.M for the sulfoximine is about 3 orders of magnitude tighter than the Km'' value of .apprx. 3 mM for L-glutamate. The previously mentioned dissociation constants obtained by enhancement titrations are also orders of magnitude tighter than Km''. L-methionine (SR)-sulfoximine may be a transition-state analogue for the glutamine synthetase reaction. The EPR spectrum (at both 9 and 35 GHz) for Mn(II) bound at the tight metal ion site is dramatically sharpened in the presence of L-methionine (SR)-sulfoximine. The EPR spectrum thus obtained is isotropic and the forbidden transitions are observed. This result is in concert with the reduced accessibility of enzyme-bound Mn(II) for H2O molecules in the presence of sulfoximine and suggests that this inhibitor is near or bound to this metal ion. The Mn(II) EPR spectrum is much more anisotropic when sulfoximine and Mg-ATP are present together and this may reflect protein conformation changes which result in ligand distortions about the Mn(II). The weak inhibitor L-methionine sulfone (K3 = 0.45 mM) and the substrate L-glutamate each produce subtle changes in the environment of bound Mn(II). More dramatic changes result from the addition of Mg-ATP to either of these 2 enzyme complexes. The changes in the EPR spectra for various complexes are seen in more detail at 35 GHz and demonstrate that most of the distortions about the Mn(II) ligand environment are rhombic in nature. These data lead to a model for the active site of glutamine synthetase in which Mn(II) at the tight metal ion site may be involved in binding the substrate L-glutamate and substrate analogue inhibitors. This binding is influenced by addition of the substrates Mg-ATP and NH4+ since progressive changes in the EPR spectra are observed. Thus the active site of the E. coli enzyme may require a full complement of substrates for catalytic function to occur and the tetrahedral geometry of methionine sulfoximine may represent a transition-state structure for the enzymic reaction.