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 [London, W. P., & Steck, T. L. (1969) Biochemistry 8, 1767-1779]. 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 . E . Mg . ATP as the subunit complex of optimum activity with Mg(II) (pHopt 7.5). With Mn(II) (pHopt 5.0), the complex of optimum activity may be either E . Mn . ATP or Mn . E . Mn . ATP, where the Mn . E complex is very tight. So that the latter two possibilities could be distinguished, titrations of the enzyme with Mn(II) were performed, electron paramagnetic resonance being used to determine free Mn(II). Four moles of Mn(II) ions was found to bind per mole of octameric enzyme, with an apparent Kd congruent to 0.54 microM. Addition of either HCl or Nd(III) ions to Chelex-treated enzyme results in the release of 3.7 +/- 0.2 Mn(II) ions. Thus, it appears that four Mn(II) are very tightly bound per octamer and that four more Mn(II) can bind tightly. Neither Mg(II) nor Ca(II) at 50 mM can displace Mn(II) from the Mn4 . 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 . E to Mn4 . E . Mn4 (+/- ATP), the intensity of the fluorescence emission of protein tryptophan residues decreases strongly and successively. The specific activities of the apo-E, Mn4 . E, and Mn4 . E . Mn4 complexes were found to be 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 congruent to 0.5-1.0 min. The total intracellular concentration of Mn(II) in ovine brain tissue was determined to be 1.9-2.6 microM, whereas the free [Mn(II)] was below 0.5 microM. Since the enzyme binds Mn(II) in preference to other divalent ions, it appears that the enzyme may exist as a manganoenzyme in vivo.

Manganese transport in eukaryotes: The role of DMT1

“…Moreover, Mn plays a role in the modulation of the immune system, and in protein, lipid and carbohydrate metabolism (Addess et al, 1997;Aschner et al, 1992;Fitsanakis et al, 2005;Malecki et al, 1999). Evidence also corroborates the involvement of Mn in the stellate process production in astrocytes (Liao et al, 2001), as well as in the metabolism of brain glutamate to glutamine, a step carried out by the astrocyte-specific enzyme, glutamine synthetase (Wedler et al, 1982;Wedler et al, 1984;Takeda, 2003).Despite its essentiality in multiple metabolic functions, Mn can be toxic at high concentrations. The brain, in particular, is highly susceptible to Mn toxicity.…”

Direct effects of manganese compounds on dopamine and its metabolite Dopac: An in vitro study

“…The recommended dietary intake for Mn is 2.3 and 1.8 mg/day for men and women, respectively. Critical enzymes, such as manganese superoxide dismutase (Mn-SOD) and glutamine synthetase, contain Mn in their structure which is essential for their functions (Wedler et al, 1982).…”

The Nudix Hydrolase Ndx1 from Thermus thermophilus HB8 Is a Diadenosine Hexaphosphate Hydrolase with a Novel Activity

“…It is suggested that Mg 2ϩ is coordinated by the glutamic acid residue, which is most likely to be Glu- 46 (42). In the proposed hydrolysis mechanism of E. coli MutT (2) and L. angustifolius Ap 4 A pyrophosphatase (29), H 2 O bound to the Mg 2ϩ ion functions as the attacking nucleophile.…”