ATP binding to purified homopolymeric plant glutamine synthetase studied by isothermal titration calorimetry

Adenosine-5′-triphosphate (ATP) binding to purified plant glutamine synthetase (GS) recombinantly overexpressed in Escherichia coli was investigated by enzyme kinetics and isothermal titration calorimetry (ITC). The concentrated enzyme was highly stable at ITC working conditions (25 °C). However, diluted preparations of the enzyme were considerably less stable but the addition of ethylene glycol to the buffer improved the long-term stability at 25 °C, although this compound precluded any possible microcalorimetric measurement. Thermodynamic parameters of binding of ATP to purified homopolymeric GS were determined both in Tris and Hepes buffer and at different ionic strength. Proton uptake by the protein was clearly detected upon ATP binding. The data obtained fitted better to a model with an n value of about one, suggesting that each enzyme subunit is able to bind a molecule of ATP. Data were also compatible with kinetic estimates of Km. We think that this kind of approach will help the structure–function characterisation of plant GS by the comparative study of wild-type and site-directed mutant polypeptides.