Oxidative turnover of soybean root glutamine synthetase. In vitro and in vivo studies

Glutamine synthetase (CS) is the key enzyme in ammonia assimilation and catalyzes the ATP-dependent condensation of NH, with glutamate to produce glutamine. CS in plants is an octameric enzyme. Recent work from our laboratory suggests that CS activity in plants may be regulated at the level of protein turnover (S.J. Temple, T.J. Knight, P.J. Unkefer, C. Sengupta-Gopalan [1993] Mol Cen Genet 236: 315-325; S.J. Temple, S. Kunjibettu, D. Roche, C. Sengupta-Gopalan [1996] Plant Physiol 112: 1723-1733; S.J. Temple, C. Sengupta-Gopalan [1997] In C.H. Foyer, W.P. Quick, eds, A Molecular Approach to Primary Metabolism in Higher Plants. Taylor & Francis, London, pp 155-177). Oxidative modification of GS has been implicated as the first step in the turnover of CS in bacteria. By incubating soybean (Glycine max) root extract enriched in GS in a metal-catalyzed oxidation system to produce the OH radical, we have shown that CS is oxidized and that oxidized CS is inactive and more susceptible to degradation than nonoxidized CS. Histidine and cysteine protect GS from metal-catalyzed inactivation, indicating that oxidation modifies the CS active site and that cysteine and histidine residues are the site of modification. Similarly, ATP and particularly ATP/glutamate give the enzyme the greatest protection against oxidative inactivation. The roots of plants fed ammonium nitrate showed a 3-fold increase in the level of GS polypeptides and activity compared with plants not fed ammonium nitrate but without a corresponding increase in the CS transcript level. This would suggest either translational or posttranslational control of GS levels.