Glutamine synthetase stability

As discussed earlier, the enzymic reaction catalyzed by glutamine synthetase requires the presence of divalent metal ions. Extensive work has been conducted on the binding of Mn2+ to the enzyme isolated from E. coli (82, 109-112). Three types of sites, each with different affinities for Mn2+, exist per dodecamer n, (12 sites, 1 per subunit) of high affinity, responsible for inducing a change from a relaxed metal ion free protein to a conformationally tightened catalytically active protein n2 (12 sites) of moderate affinity, involved in active site activation via a metal-ATP complex and n3 (48 sites) of low affinity unnecessary for catalysis, but perhaps involved in overall enzyme stability. The state of adenylylation and pH value alter the metal ion specificity and affinities. 

The main role of the IIA cations in the activation of enzymes seems to be that of weak Lewis acids. In addition the cation may serve as a template to bridge enzyme and substrate and bring them into the correct relative orientation for reaction. Furthermore these cations may stabilize or produce certain protein conformations. Thus glutamine synthetase binds 24 moles of Mn2+ per mole of protein. The binding of the first 12 cations results in conformational changes that lead to the formation of 12 new sites for the binding of the remaining 12 cations, which then have a catalytic role. 

Wedler f al. (1978) have identified two forms of glutamine synthetase in Bacillus caldolytiens and Darrow and Knotts (1977) have shown two forms in Rhizobium japonicum and other free living root nodule bacteria. In both cases the two forms differ in their isoelectric points and stability. The work of Darrow and Knotts (1977) indicates that the two forms are not the result of differences in adenylation state of a single form. Type I appears similar to the E. coli enzyme in stability and in being susceptible to adenylation. Type II however is not adenylated and is more unstable. 

The pea leaf glutamine synthetase appears less stable than the pea seed enzyme (O Neal and Joy, 1973b). These workers found that Mg " " and Mn + increased stability of crude and partly purified enzyme, Mn " " being more effective than Mg + especially at low pH. Stability was further improved by addition of sucrose (0.5-1.5 M), fructose (1.3-2.4 M), or ethylene glycol (10-40%). Highly purified enzyme was more stable without Mg " " if molar sucrose or 20-30% ethylene glycol was present. Mercaptoethanol enhanced stability at high (35°-50"C) but not low (0 -3 C) temperature in the presence of Mg + but decreased activity in the presence of Mn " ", except in the presence of ATP. ATP at concentrations which can complex all the divalent cations decreases stability but if Mg or Mn + are present in excess ATP increases stability. 

---