Functional characterization of Legionella pneumophila Cu+ transport ATPase. The activation by Cu+ and ATP

PLACENTI, M. AGUEDA; ROMAN, ERNESTO A.,; GONZÁLEZ FLECHA, F. LUIS; GONZÁLEZ-LEBRERO, RODOLFO M.

BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 2022

0005-2736

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 ∼37ºC 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  ~30000.  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]→∞).