Bacterial Glutamine Synthetase Research Articles

27] Glutamine synthetase from mammalian tissues

This chapter provides an overview of the glutamine synthetase from mammalian tissues. The glutamine synthesis reaction is freely reversible. When the enzyme is incubated with 10 mM concentrations each of L-glutamate, ammonium ions, and ATP in the presence of Mg2+ at pH 7.0 and 37°, equilibrium is attained when about 90% of the L-glutamate is converted to L-glutamine. Substitution of hydroxylamine for ammonia in this system leads to a reaction that goes to greater than 99% of completion. The glutamine synthetases of mammalian origin closely resemble each other with respect to amino acid composition, subunit structure, and molecular weight. They differ substantially in these respects from bacterial glutamine synthetases, some of which exist in adenylylated forms. Glutamine synthetase activity may be followed by measuring the rate of formation of inorganic phosphate, ADP, or glutamine. ADP may be determined by coupling the glutamine synthetase reaction with those catalyzed by pyruvate kinase and lactate dehydrogenase. By use of reaction mixtures containing labeled glutamate, the disappearance of glutamate and the formation of glutamine may be determined. A commonly used procedure for determining glutamine synthetase activity involves replacement of ammonia by hydroxylamine. In this reaction, γ-glutamylhydroxamate is formed, which gives a characteristic color reaction on addition of ferric chloride.

Adenylylation of Bacterial Glutamine Synthetase: Physiological Significance

This chapter focuses on the adenylylation of bacterial glutamine synthetase. The enzyme glutamine synthetase catalyzes the synthesis of glutamine from ammonia and glutamate in an ATP-dependent reaction. In bacteria and higher plants, it also participates in net synthesis of glutamate from ammonia and 2-oxoglutarate by functioning in a cycle with glutamate synthase. Physiological studies of Hölzer and colleagues indicated that the GS of Escherichia coli was very rapidly adenylylated when the organism was shifted from N-limited medium to a medium containing excess ammonium. Strains of S. typhimurium (glnE) that lack the ability to adenlylate GS show a large growth defect upon shift from N-limited media, media in which they have accumulated high levels of GS, to media containing a high concentration of NH 4 +. This growth defect appears to be because of excessive GS activity after the shift because it is eliminated in glnE strains that also synthesize abnormally low amounts of GS in both N-limited and NH4+, it is prolonged in glnE strains that also synthesize abnormally high amounts of GS in NH4+ excess media and the latter strains continue to show a growth defect even after adaptation to NH 4 +, and glnE strains have normal amounts of both of the glutamatebiosynthetic enzymes under N-limited conditions and after NH 4 + upshift. At present, there appears to be no simple explanation for the distribution of this covalent modification. Although adenylylation of GS occurs widely among gram-negative bacteria, a more complete understanding of the physiological requirements for adenylylation of GS will contribute to understanding the basis for distribution of this covalent modification among the prokaryotes.

Covalent modification of bacterial glutamine synthetase: physiological significance.

Stadtman, Holzer and their colleagues (reviewed in Stadtman and Ginsburg 1974) demonstrated that the enzyme glutamine synthetase (GS) [(L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2] is covalently modified by adenylylation in a variety of bacterial genera and that the modification is reversible. These studies further indicated that adenylylated GS is the less active form in vitro. To assess the physiological significance of adenylylation of GS we have determined the growth defects of mutant strains (glnE) of S. typhimurium that are unable to modify GS and we have determined the basis for these growth defects. The glnE strains, which lack GS adenylyl transferase activity (ATP: [L-glutamate: ammonia ligase (ADP-forming)] adenylyltransferase, EC 2.7.7.42), show a large growth defect specifically upon shift from a nitrogen-limited growth medium to medium containing excess ammonium (NH4+). The growth defect appears to be due to very high catalytic activity of GS after shift, which lowers the intracellular glutamate pool to approximately 10% that under preshift conditions. Consistent with this view, recovery of a rapid growth rate on NH4+ is accompanied by an increase in the glutamate pool. The glnE strains have normal ATP pools after shift. They synthesize very large amounts of glutamine and excrete glutamine into the medium, but excess glutamine does not seem to inhibit growth. We hypothesize that a major function for adenylylation of bacterial GS is to protect the cellular glutamate pool upon shift to NH4+ -excess conditions and thereby to allow rapid growth.

Glutamine synthetase from Mycobacterium avium.

Mycobacterium avium was previously shown to be dependent upon ammonia or glutamine as a nitrogen source. In an effort to assess the physiology of ammonia assimilation by M. avium, a characterization of its glutamine synthetase was performed. The enzyme from M. avium was purified by streptomycin sulfate treatment, ammonium sulfate precipitation, and affinity chromatography. The enzyme was unusual in that it had a pH optimum of 6.4 and maximum enzyme activity was obtained between 50 and 60 degrees C as shown by the transferase assay. The glutamine synthetase activity from batch-cultured cells decreased with increasing concentration of ammonium chloride in the range of 0.25-5 mumol/mL of medium, which demonstrated a response to environmental supply of a nitrogen source. The mycobacterial enzyme was similar to the other bacterial glutamine synthetases in terms of molecular weight and sedimentation coefficient which were 600 000 and 19.5 S, respectively, and enzyme activity was lost by treatment with a glutamate analog, methionine sulfoximine. The isoelectric point was, however, pH 4.5. Treatment of the enzyme with snake venom phosphodiesterase resulted in an increase in specific activity. AMP was released by the phosphodiesterase treatment, thus demonstrating that M. avium glutamine synthetase was regulated by adenylylation modification.

Oxidative modification of glutamine synthetase. II. Characterization of the ascorbate model system.

The first step in the proteolytic degradation of bacterial glutamine synthetase is a mixed function oxidation of one of the 16 histidine residues in the glutamine synthetase subunit (Levine, R.L. (1983) J. Biol. Chem. 258, 11823-11827). A model system, consisting of oxygen, a metal ion, and ascorbic acid, mimics the bacterial system in mediating the oxidative modification of glutamine synthetase. This model system was studied to gain an understanding of the mechanism of oxidation and of factors which control the susceptibility of the enzyme to oxidation. Availability of substrates and the extent of covalent modification of the enzyme (adenylylation) interact to modulate susceptibility of the enzyme to oxidation. This interaction provides the biochemical basis for physiologic regulation of intracellular proteolysis of glutamine synthetase. The oxidative modification requires hydrogen peroxide. While the reaction may involve Fenton chemistry, the participation of free radicals, superoxide anion, and singlet oxygen could not be demonstrated.

Comparative biochemical and immunological studies of bacterial glutamine synthetases.

Antisera prepared against adenylylated and unadenylylated Escherichia coli glutamine synthetase cross-reacted with the glutamine synthetases from a number of gram-negative bacteria and one gram-variable species as demonstrated by immunodiffusion and inhibition of enzyme activity. In contrast, the antisera did not cross-react with the glutamine synthetases from gram-positive bacteria (with one exception) nor with the synthetases of higher organisms. Modification of the various glutamine synthetases by covalent attachment of adenosine 5'-monophosphate (or other nucleotides) was tested for by determining whether or not snake venom phosphodiesterase altered catalytic activity in a manner similar to its effect on adenylylated E. coli glutamine synthetase. Only the activity of the glutamine synthetases from gram-negative bacteria grown with specific levels of nitrogen sources could be altered by snake venom phosphodiesterase. In addition, a relative order of antigenic homology between cross-reacting enzymes was suggested based on the patterns of spur formation in the immunodiffusion assay.