Glutamine synthetase adenylation

Thus, we have determined the distances between the adenylyl moiety and the two divalent metal ion binding sites on glutamine synthetase by 13C and 3 P NMR, spin-labeled EPR, and fluorescence energy transfer methods. The results obtained from each method are in good agreement. The data show that the adenylyl regulatory site is close to the catalytic site (12-20 A). Additional data on the rotational correlation time of the adenyl derivatives reveal that the adenylyl site is located on the surface of the enzyme. 

The adenylation and phosphorolysis reactions are catalyzed by the same enzyme, adenylyl transferase. Sequence analysis indicates that this adenylyl transferase comprises two homologous halves, suggesting that one half catalyzes the adenylation reaction and the other half the phospholytic de-adenylation reaction. What determines whether an AMP unit is added or removed The specificity of adenylyl transferase is controlled by a regulatory protein (designated P or Pjj), a trimeric protein that can exist in two forms, P and Pq (Figure 24.27). The complex of P and adenylyl transferase catalyzes the attachment of an AMP unit to glutamine synthetase, which reduces its activity. Conversely, the complex of Pj) and adenylyl transferase removes AMP from the adenylylated enzyme. 

Figure 24.26. Regulation by Adenylation. (A) A specific tyrosine residue in each subunit in glutamine synthetase is modified by adenylation. (B) Adenylation of tyrosine is catalyzed by a complex of adenylyl transferase (AT) and one form of a regulatory protein (P ). The same enzyme catalyzes deadenylation when it is complexed with the other form (Pq) of the regulatory protein.

Glutamine synthetase is a dodecamer composed of 12 identical subunits. Each subunit can be adenylated. How many quantita-... 

Recent primary structure sequence studies on Escherichia colt glutamine synthetase demonstrated that the covalently bound active site adenylic acid residue is attached to a tyrosine residue (235). The sequence of amino acids around the derivated tyrosine residue is ... 

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. 

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 glutamine synthetase of E. coli is partially inhibited by adenylate, being maximally inhibited only in the presence of products of eight different glutamine-utilizing pathways. The interpretation of these results is complicated b> the more recent discovery of two forms of glutamine synthetase in this organism and of the two enzymes required for their interconversion (54)- The sheep brain enzyme is not subject to these kinds of control mechanism (66). 

Fig. 39.6. Enzyme activity as a function of lanthanide ionic radius. Filled circles relative biosynthetic activity of adenylated glutamine synthetase, data from Wedler and D Aurora (1974). Open circles reciprocal half-times for the conversion of trypsinogen to trypsin, data from Gomez et al. (1974).

Other forms of regulatory phosphorylations have been documented as well. For example, DNA transcription and replication may be regulated by phosphorylation of histones on serine, histidine, or, lysine residues (Isen-berg, 1979 Chen et al, 1974). Proteins with phosphotyrosine amino acids have often been found in the viral transformation processes (Hayman, 1981 Martensen, 1982). Also, 5 -adenylyl-0-tyrosine has been isolated as the known regulator in the adenylation of glutamine synthetase (Shapiro and Stadtman, 1968). 

The activity of glutamine synthetase from E. coli is regulated mainly by covalent adenylation of a specific hydroxyl group of a tyrosine moiety. Thus it has been possible to measure the distance from the catalytic Mn + or Co + ion to the phosphorus atom of the AMP moiety bound to the protein (Villafranca et al, 1978). This distance was 7 A. Moreover, it was shown that the phosphoryl moiety is immobilized on the surface of the protein but the adenyl part of the AMP group has considerable flexibility. 

The supply of nonnucleotide substrates may also be an important control mechanism for these pathways. In Ehrlich ascites tumor cells aspartate concentrations were found to be rate-limiting for adenylate synthesis from hypoxanthine (46), and glutamine levels limited guanylate synthetase activities in these cells and in erythrocytes (47)-... 

The glutamine analogue, diazo-oxo-norleucine, and the aspartate analogue, hadacidin (iV-formyl hydroxyaminoacetic acid), inhibit guanylate synthetase and adenylosuccinate synthetase, respectively. Alanosine (2-amino-3-nitrohydroxylaminopropionic acid) is also an inhibitor of adenylate synthesis from inosinate, but its mechanism of inhibition is not yet clear (52). 

Phenylacetate, after conversion to the coenzyme A (in the presence of ATP) derivative (phenylacetyl CoA), is coupled with glutamine to yield phenylace-tylglutamine. The product of the reaction is found in normal urine. The enzyme catalyzing the reaction is present in liver and kidney. The reaction is assumed to develop in three steps. First, the enzyme reacts with phenylacetic acid and ATP to yield enzyme-bound phenylacetyl adenylate and pyrophosphate. Second, the enzyme-bound phenylacetyl adenylate reacts with reduced coenzyme A to yield free phenylacetyl-S-CoA, AMP, and the free enzyme. Third, phenylacetyl CoA reacts with glutamine and a phenylacetylglutamine synthetase to yield the final product. 

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