Glutamine synthetase from Salmonella typhimurium: Manganese(II), substrate, and inhibitor interaction with the unadenylylated enzyme

Abstract Unadenylylated glutamine synthetase (EC 6.3.1.2) was isolated and purified to homogeneity from Salmonella typhimurium . The enzyme molecule is a symmetrical aggregate of 12 subunits arranged in two hexagonal layers, as is evident from electron micrographs. The subunit molecular weight of the enzyme was found to be approximately 50,000 by polyacrylamide gel electrophoresis in sodium dodecyl sulfate when compared to Escherichia coli glutamine synthetase and other protein standards. A long tube of glutamine synthetase was formed as a single-stranded coil resulting from incubation of the enzyme in a low ionic strength buffer. A study of Mn(II) binding to the unadenylylated enzyme at 25 °C was conducted as a function of pH. At pH 7.1 two classes of metal ion sites per subunit were found with K D values of 3.7 × 10 −6 and 1.7 × 10 −4 m , while at pH 6.8 these values were 1.1 × 10 −5 and 1.0 × 10 −4 m , respectively. Only one set of binding sites was observed at pH 6.2 with a K D value of 1.0 × 10 −4 m . The metal ion binding sites were further investigated by monitoring proton relaxation rates (prr) and the epr spectrum of enzyme-bound Mn(II). The longitudinal prr of water protons at pH 7.1 indicate that protons interacting with enzyme-Mn(II) at the “tight” site ( K D = 3.7 × 10 −6 ) are de-enhanced ( ϵ b 1 = 0.42) and result from water protons beyond the inner coordination sphere. The second Mn(II) site has a value of ϵ b 2 = 35 for the binary enhancement, suggesting that this site probably has two to three rapidly exchanging water molecules in its coordination sphere. The epr spectrum of enzyme-bound Mn(II) at the “tight” site is isotropic and is dramatically sharpened by adding the substrate analog methionine sulfoximine. Subsequent addition of ATP or the ATP analog, AMP-PCP (adenylyl methylene diphosphate) produced anisotropic spectra that were similar, suggesting that both ATP and AMP-PCP bind similarly on the enzyme surface. However, a marked change in the Mn(II) environment from anisotropic to near cubic results from the addition of ADP to the quaternary enzyme-Mn(II)-sulfoximine- (AMP-PCP) complex, indicating that ADP displaces AMP-PCP. No change in the anisotropic spectrum due to the enzyme-Mn(II)-sulfoximine-ATP complex is seen by the addition of ADP. This experimental result supports the experimental findings of Ronzio and Meister [ Proc. Nat. Acad. Sci. USA 59 , 164 (1968)], who established that ATP phosphorylates methionine sulfoximine, thereby producing an inactive enzyme. The allosteric effectors, AMP and Trp, have little effect on the epr spectrum of the complex formed from Mn(II), enzyme, sulfoximine, and ADP, suggesting the absence of direct coordination of AMP or Trp to the bound Mn(II). The prr and epr results reported herein with glutamine synthetase from S. typhimurium when compared to those seen for the enzyme from E. coli [Villafranca et al., Biochemistry 15 , 544 (1976)] demonstrate some similarities but also many substantial differences between the enzymes from these two bacterial sources.