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The conversion of native adenylylated glutamine synthetase into phosphotyrosine enzyme by micrococcal nuclease
Abstract:
Micrococcal nuclease treatment of the native adenylylated glutamine synthetase from M. smegmatis yielded adenosine and phosphotyrosyl enzyme. The rate of the deadenosylation reaction was monitored by the appearance of the adenosine in HPLC analysis. The o-phosphotyrosyl enzyme had catalytic activity comparable to that of the adenylylated enzyme suggesting that the adenosine part in AMP was not essential to the regulation of the enzyme activity. Further, upon treatment of the phosphotyrosyl enzyme with alkaline phosphatase, the glutamine synthetase activity was increased. This means that the regulation site of glutamine synthetase by covalent modification simply requires the phosphorylation of the tyrosine residue.
Insights
Micrococcal nuclease treatment of glutamine synthetase revealed that phosphorylation of tyrosine is key for enzyme regulation. Adenosine modification is not essential for controlling glutamine synthetase activity.
Area of Science:
- Biochemistry
- Enzymology
- Microbiology
Background:
- Glutamine synthetase (GS) is a crucial enzyme in nitrogen metabolism.
- Covalent modification, specifically adenylylation, regulates GS activity in many bacteria.
- The role of adenylylation in M. smegmatis GS regulation requires further elucidation.
Purpose of the Study:
- To investigate the role of adenylylation and phosphorylation in the regulation of M. smegmatis glutamine synthetase.
- To determine if the adenylyl moiety is essential for enzyme activity regulation.
Main Methods:
- Treatment of native adenylylated M. smegmatis GS with micrococcal nuclease.
- Monitoring deadenylation using High-Performance Liquid Chromatography (HPLC).
- Assessing catalytic activity of phosphotyrosyl enzyme and effect of alkaline phosphatase treatment.
Main Results:
- Micrococcal nuclease treatment released adenosine and generated phosphotyrosyl enzyme.
- The phosphotyrosyl enzyme exhibited catalytic activity similar to the adenylylated form.
- Dephosphorylation of the phosphotyrosyl enzyme by alkaline phosphatase increased glutamine synthetase activity.
Conclusions:
- The adenylyl moiety of AMP is not essential for regulating M. smegmatis glutamine synthetase activity.
- Regulation of M. smegmatis glutamine synthetase by covalent modification primarily depends on tyrosine residue phosphorylation.