o-Phosphotyrosyl glutamine synthetase: modification of the nucleotide ligation site of adenylylated glutamine synthetase.
The nucleotide ligation site of adenylylated glutamine synthetase, which contains a unique tyrosyl residue linked through a phosphodiester bond to 5'-AMP, was studied by digestion with three hydrolytic enzymes. The products on micrococcal nuclease digestion were adenosine and o-phosphotyrosyl glutamine synthetase. The Km for this macromolecular substrate with the nuclease was 40 microM, at pH 8.9. The glutamine synthetase activity was not affected by deadenosylation with the nuclease, in contrast to SVPDE digestion, with which the glutamine synthetase activity was markedly increased. The Km for the native adenylylated glutamine synthetase with the SVPDE was 36 microM, i.e., similar to that for the nuclease. When the isolated o-phosphotyrosyl enzyme was incubated with alkaline phosphatase at pH 7.2, the glutamine synthetase activity rapidly increased to the same level as that of the SVPDE treated enzyme. Furthermore, kinetic properties of the o-phosphotyrosyl glutamine synthetase were compared with those of the adenylylated enzyme. The optimum pH, apparent Km for each of three substrates, glutamate, ATP, and NH3, and Vmax were in good agreement, as to either Mg2+- or Mn2+-dependent biosynthetic activity. From these results we can conclude that the regulation of glutamine synthetase activity simply requires the phosphorylation of the tyrosyl residue in each subunit, without recourse to adenylylation.
Kimura
K
K
Laboratory of Biochemistry, College of Science, Rikkyo (St. Paul's) University, Tokyo.
Nakano
Y
Y
Matsuoka
K
K
D000241:Adenosine; D000469:Alkaline Phosphatase / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism*; D008836:Micrococcal Nuclease / Q000378:metabolism; D009161:Mycobacterium / Q000201:enzymology; D019000:Phosphotyrosine; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D019000:Phosphotyrosine; D014443:Tyrosine; D000469:Alkaline Phosphatase; D008836:Micrococcal Nuclease; D005974:Glutamate-Ammonia Ligase; D000241:Adenosine