ATPase enzymes that transport heavy metal ions (Cu, Ag, Zn, Cd, Pb, Co) belong to the P1B-type. This class is prevalent in all kingdoms of life and uses energy provided by ATP hydrolysis to pump ions across cell membranes against thermodynamic gradients. Cu-ATPases transport the nutrient trace metal copper specifically as CuI and play essential roles in activation of copper enzymes and in removal of excess copper from cell cytosols. Malfunctions in these copper pumps are associated with copper imbalance diseases in humans including Menkes and Wilson diseases and with a series of neurodegenerative conditions.[1]
Cu-ATPases in prokaryotes have the general name 'CopA'. They remove excess copper from the cytosol, utilising the Gibbs free energy of ATP hydrolysis to transport Cu(I) ions across membranes. Hence the enzymatic activity can be estimated by monitoring hydrolysis of ATP directly.A widely-used protocol is based upon colourimetric determination of product inorganic phosphate. This approach is subject to high backgrounds due to extraneous sources of phosphate and to non-enzymatic ATP hydrolysis occurring at the colour development stage.
ATP hydrolysis may be monitored, without the above complications, by simultaneous detection of ATP depletion and ADP accumulation via 1H-NMR [2]. The technique has been developed here to allow continuous and automated monitoring of activity in real-time. Importantly, it does not require additional reagents. The use of cryogenic probes further increases sensitivity, enabling detection of as little as 10 µg of enzyme. The approach provides a significant advantage as membrane copper pumps are difficult to express and isolate in useful yields.
References
[1] C.Rensing, et al., Proc. Natl. Acad. Sci 2000, 97, 652
[2] B. Guo, et al., Chem. Com 2014, 50, 12037-1203.