[No authors listed]
The thermophilic, sulfur metabolizing Archaeoglobus fulgidus contains two genes, AF0473 and AF0152, encoding for PIB-type heavy metal transport ATPases. In this study, we describe the cloning, heterologous expression, purification, and functional characterization of one of these ATPases, CopA (NCB accession number AAB90763), encoded by AF0473. CopA is active at high temperatures (75 degrees C; E(a) = 103 kJ/mol) and inactive at 37 degrees C. It is activated by Ag+ (ATPase V(max) = 14.82 micromol/mg/h) and to a lesser extent by Cu+ (ATPase V(max) = 3.66 micromol/mg/h). However, Cu+ interacts with the enzyme with higher apparent affinity (ATPase stimulation, Ag+ K(12) = 29.4 microm; Cu+ K(12) = 2.1 microm). This activation by Ag+ or Cu+ is dependent on the presence of millimolar amounts of cysteine. In the presence of ATP, these metals drive the formation of an acid-stable phosphoenzyme with apparent affinities similar to those observed in the ATPase activity determinations (Ag+, K(12) = 23.0 microm; Cu+, K(12) = 3.9 microm). However, comparable levels of phosphoenzyme are reached in the presence of both cations (Ag+, 1.40 nmol/mg; Cu+, 1.08 nmol/mg). The stimulation of phosphorylation by the cations suggests that CopA drives the outward movement of the metal. CopA presents additional functional characteristics similar to other P-type ATPases. ATP interacts with the enzyme with two apparent affinities (ATPase K(m) = 0.25 mm; phosphorylation K(m) = 4.81 microm), and the presence of vanadate leads to enzyme inactivation (IC(50) = 24 microm). This is the first Ag+/Cu+ -ATPase expressed and purified in a functional form. Thus, it provides a model for structure-functional studies of these transporters. Moreover, its characterization will also contribute to an understanding of thermophilic ion transporters.
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