Evidence for methionine sulfoximine as a transition-state analog for glutamine synthetase from NMR and EPR data

Abstract

Manganese(II)bound at the “tight” metal ion site of unadenylylated glutamine synthetase (E. coli W) has two rapidly exchanging first coordination sphere water molecules. The solvation number was evaluated from a study of the frequency dependence of 1 pT 1p , the paramagnetic contribution to the longitudinal relaxation rate of solvent protons. The number of rapidly exchanging water molecules is reduced to one in the presence of saturating L-glutamate and to ∼0.2 when L-methionine SR-sulfoximine (MSOX) is present. MSOX is a linear competitive inhibitor (K I=3μM) of glutamine synthetase when L-Glu is the substrate. The dissociation constant of MSOX measured by following the 18 fold decrease in 1 pT 1p (at 48 MHz) is 30μM and is lowered to ∼9μM in the presence of ADP. The high affinity of MSOX for the enzyme suggests that this compound mimicks the “transition-state” for the glutamine synthetase reaction. Further evidence for this postulate is found from the dramatic sharpening of the epr spectrum of enzyme-bound Mn(II) in the presence of MSOX and MSOX plus ADP. The intense change in the epr spectrum arises from reduced solvent accessibility to bound Mn(II) and conformational changes produced by binding MSOX and ADP. The suggestion is made from these data that L-Glu and MSOX bind near or directly to the Mn(II) at the “tight” metal ion site in glutamine synthetase isolated from E. coli W.