Glutamine synthetase from ovine brain is a manganese(II) enzyme

Abstract

The activation of ovine brain glutamine synthetase by Mn(II) or Mg(II) was studied by steady-state kinetics. The metal ion concentration was varied at several fixed concentrations of ATP, and vice versa, and the resultant kinetic curves were analyzed according to the method of London and Steck. The data for Mg(II) indicated optimal activation at Mg:ATP = 2:1, whereas that for Mn(II) occurred at Mn:ATP = 1:1. This was interpreted as indicating formation of Mg.cntdot.E.cntdot.Mg.cntdot.ATP as the subunit complex of optimum activity with Mg(II) (pHopt 7.5). With Mn(II) (pHopt 5.), the complex of optimum activity may be either E.cntdot.Mn.cntdot.ATP or Mn.cntdot.E.cntdot.Mn.cntdot.ATP, where the Mn.cntdot.E complex is very tight. So that the latter 2 possibilities could be distinguished, titrations of the enzyme with Mn(II) were performed, electron paramagnetic resonance being used to determine free Mn(II). Mn(II) ions (4 mol) bound to 1 mol of octameric enzyme, with an apparent Kd .simeq. 0.54 .mu.M. Addition of either HCl or Nd(III) ions to Chelex-treated enzyme results in the release of 3.7 .+-. 0.2 Mn(II) ions. It appears that 4 Mn(II) are very tightly bound per octamer and that 4 more Mn(II) can bind tightly. Neither Mg(II) nor Ca(II) at 50 mM can displace Mn(II) from the Mn4.cntdot.E complex, but Mn(II) still binds tightly to apoenzyme or Mn4E in the presence of 50 mM Mg(II). As one proceeds from apo-E to Mn4.cntdot.E to Mn4.cntdot.E.cntdot.Mn4 (.+-. ATP), the intensity of the fluorescence emission of protein tryptophan residues decreases strongly and successively. The specific activities of the apo-E, Mn4.cntdot.E and Mn4.cntdot.E.cntdot.Mn4 complexes were 0, 50 and 200 units/mg, respectively. If apoenzyme is added to a continuous coupled assay system with Mg(II), buffer, and with or without Mn(II) present, a time-dependent activation is observable with t1/2 .simeq. 0.5-1.0 min. The total intracellular concentration of Mn(II) in ovine brain tissue was 1.9-2.6 .mu.M, whereas the free [Mn(II)] was 0.5 .mu.M. Since the enzyme binds Mn(II) in preference to other divalent ions, it appears that the enzyme may exist as a manganoenzyme in vivo.