Publikationsrepositorium - Helmholtz-Zentrum Dresden-Rossendorf

The aim of this study is to investigate the structure and function of P-type-ATPases. Enterococcus hirae is a gram-positive lactic acid bacterium with two copper ATPases CopA and CopB. They show sequence similarity to known P-type-ATPases. The monovalent copper exporting ATPase CopB is a central regulator for copper homeostasis in E. hirae which shows 39 % sequence similarity to the sarcoplamic reticulum calcium ATPase SERCA1a of rabbit hind leg muscle (Oryctolagus cuniculus). SERCA1a undergoes large conformational changes of cytoplasmatic and transmembrane domains to translocate ions. Despite some former work, the transport of copper and the biochemical properties of the ATPase, however, has to be analyzed and the observation of hydrolytic activities has to be pursued. During thesis work the functional status of conformational states was studied by spectroscopy work on metal and nucleotide binding. The ability of nonionic detergents to stabilize the membrane-bound enzymes was used to work in lipid analogue environment, whereby the effect of light scattering of lipid systems is prevented. I have investigated the secondary structure of purified CopB in the absence and presence of the non-hydrolyzable ATP analogs ATPgS, mantATP and silver (a redox inert Cu+ analog) using circular dichroism spectroscopy. Binding of metals unfolds the protein, whereas ATP analogs partially elliminate this effect. ATPgS and silver form an optically active complex. Negative and positive CD peaks appear, at 257 nm and 277 nm, respectively, at a ratio of 1:3 of Ag:ATPgS corresponding to the predominant species ATPgS3Ag4. CopB competes with complex formation by binding both ATPgS and silver. To my knowledge this is the first description of such a complex. It is used in this work as a possible high sensitive realtime ATPase monitor. This assay could ultimately be exploited to determine binding affinities of nucleotide, silver and CopB in enzyme assays in real time. In the present work it is used to determine binding affinitiy of CopB to ATPgS. In addition to check the influence of the binding of ATP analogs and silver on stability of CopB, the protein was denaturated in the absence and presence of ATPgS, mantATP, ADP and silver. Whereas ATPgS and mantATP stabilize CopB, the same nucleotide-CopB complex is destabilized by silver. This evidences a strong negative coupling between the nucleotide and metal binding site as an important output of my work.