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Cu+-ATPases are integral membrane proteins belonging to the IB subfamily of the P-type ATPases that couple Cu+ transport to the hydrolysis of ATP. As some structural and functional particularities arise for Cu+-ATPases, several authors suggest that some of the reaction steps of the Albers-Post model postulated for other P-ATPases may be different. In this work we describe a functional characterization of Legionella pneumophila Cu+-ATPase (LpCopA), the first PIB-ATPase whose structure was determined by X-ray crystallography. Cu+-ATPase activity of the enzyme presents a maximum at ∼37 °C and pH 6.6–6.8. Phospholipids enhance LpCopA Cu+-ATPase activity in a non-essential mode where optimal activity is achieved at an asolectin molar fraction of 0.15 and an amphiphile-protein ratio of ~30,000. As described for other P-ATPases, Mg2+ acts as an essential activator. Furthermore, Cu+-ATPase activity dependence on [Cu+] and [ATP] can both be described by a sum of two hyperbolic functions. Based on that, and the [Cu+] and [ATP] dependencies of the best fitting parameters of the hyperbolae pointed above, we propose a minimal reaction scheme for the catalytic mechanism that shares the basic reaction steps of the Albers-Post model for P-type ATPases. The reaction scheme postulated contemplates two different binding affinities for a single ATP (apparent affinities of 0.66 and 550 μM at [Cu+] → ∞) and, binding of at least, 2 Cu+ with different affinities as well (apparent affinities of 1.4 and 102.5 μM at [ATP] → ∞). |
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