Glutamine synthetase nucleotides

There are two cation-binding sites per subunit, classified nL and n2. The n, site is a structural site, which may involve the reorientation of a glutamate carboxyl group. Metals bind first at this site. The n2 site is the catalytic site and may bind metal or metal-nucleotide. Co111 and Cr111 can be incorporated into the nj metal-binding sites in un-adenylylated glutamine synthetase from E. co/i.318 Both derivatives were inactive, but were able to bind Mn2+ at the n2 site. Comparison of the quaternary enzyme-Crin-Mnn-ADP (which shows spin-spin interaction between the two metal centres) with enzyme-Com-Mnn-ADP leads to an estimate of the distance between n, and n2 sites of 7 2 A. 

Lightfoot, D.A., Green, N.K. Cullimore, J.V. (1988). The chloroplast-located glutamine synthetase of Phaseolus vulgaris L. Nucleotide sequence, expression in different organs and uptake into isolated chloroplasts. Plant Molecular Biology 11, 191-202. 

This reaction requires catalytic amounts of nucleotide. Activity in the transferase assay generally gives rates several times higher than those obtained in the synthetase assays [Eqs. (1) and (2)] and for this reason is often used for measurement of glutamine synthetase, especially in relatively crude preparations. However caution should be exercised in the use of the transferase assay as a similar reactitm can also be catalyzed by some glutaminases and certain other amidases (Meister et al., 1955), although in these cases the reaction is not dependent on ADP, phosphate, or divalent metal cation. Levintow et al. (1955) demonstrated that the synthetase activity and the ADP phosphate divalent cation dependent transferase activity are functions of the same enzyme. 

Interpretation of bacterial glutamine synthetase is further complicated due to the enzyme existing in both adenylated and deadenylated forms. Whereas only the deadenylated form is biosynthetically active both forms catalyze transferase activity. Glutamine synthetase can also catalyze the arsenolysis of glutamine [Eq. (4)]. When the enzyme is incubated with L-glutamine, catalytic amounts of nucleotide and divalent metal ions, glutamine is converted to glutamate and ammonia. 

Nucleotides have also been shown to inhibit glutamine synthetase from other sources, notably ADP, 5 -AMP, and CTP inhibit the enzyme from soybean root nodules (McParland et al., 1976), 5 -AMP and CTP that from E. coli (Woolfolk and Stadtman, 1967), B. subtilis (Deuel and Stadtman, 1970), and N. crassa (Kapoor and Bray, 1968), and 5 -AMP and CMP inhibit the Chlorella enzyme (Evstigneeva, et al., 1974). In contrast the rat liver and ovine brain enzymes are not appreciably inhibited by these nucleotides (Meister, 1974). When testing for inhibition by nucleotides it is necessary to have sufficient divalent cation present to ensure that inhibition is not simply a result of inadequate divalent cations due to chelation by the nucleotide. The variation in inhibition by nucleotides is dependent to some degree on the source of divalent metal cation thus O Neal and Joy (1975) found ADP and 5 -AMP to be more inhibitory to the Mg +-compared with the Mn +-dependent activity. 5 -AMP was also found to be more inhibitory to the magnesium dependent activity of B. subtilis enzyme (Deuel and Stadtman, 1970). 

Remarkable differences can also be found within a single species. For example, the glutamine synthetase from rat kidney acts on its substrate ten times faster than does the analogous enzyme from rat muscle (Iqbal and Ottaway, 1970). Again, cancer cells maintain a more rapid cell cycle than normal cells hence they are more sensitive to drugs which interfere with the synthesis of nucleotides. 

Fig. S. Depiction of the active site of Escherichia coli glutamine synthetase showing the n, and 2 metal-ion sites and the probable location of the nucleotide when Cr + (A) or Mn + (B) is the divalent cation.

Figure 20.9 The positions in the pathway for de novo pyrimidine nucleotide synthesis where GLUCOSE provides the ribose molecule and GLUTAMINE provides nitrogen atoms. Glucose forms ribose 5-phosphate, via the pentose phosphate pathway (see chapter 6), which enters the pathway, after phosphorylation, as 5-phospho-ribosyl 1-pyrophosphate. Glutamine provides the nitrogen atom to synthesise carbamoylphos-phate (with formation of glutamate), and also to form cytidine triphosphate (CTP) from uridine triphosphate (UTP), catalysed by the enzyme CTP synthetase. It is the amide nitrogen of glutamine that is the nitrogen atom that is provided in these reactions.

Cytidine triphosphate (CTP) is produced by amination of UTP by CTP synthetase (Figure 22.22). [Note The nitrogen is provided by glutamine—another example of a reaction in nucleotide biosynthesis in which this amino acid is required.]... 

The atoms of the pyrimidine ring are derived from carbamoyl phosphate and aspartate, as shown in Fig. 15-14. The de novo biosynthesis of pyrimidine nucleotides is shown in Fig. 15-15. The first completely formed pyrimidine ring is that of dihydroorotate. Only after oxidation to orotate is the ribose attached to produce orotidylate. The compound 5-phosphoribosyl 1-pyrophosphate (P-Rib-PP) provides the ribose phosphate. L-Glutamine is used as a substrate donating nitrogen atoms at reactions 1 and 9, catalyzed by carbamoyl phosphate synthetase II and CTP synthetase, respectively a second... 

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