INVESTIGADORES
GONZALEZ FLECHA Francisco Luis
artículos
Título:
Activation of Archaeoglobus fulgidus Cu(+)-ATPase CopA by cysteine
Autor/es:
YING YANG; ATIN K. MANDAL; LUIS M. BREDESTON; F. LUIS GONZÁLEZ FLECHA; JOSÉ M. ARGÜELLO
Revista:
BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES
Editorial:
Elsevier
Referencias:
Año: 2007 vol. 1768 p. 495 - 501
ISSN:
0005-2736
Resumen:
CopA, a thermophilic ATPase from Archaeoglobus fulgidus, drives the
outward movement of Cu(+) across the cell membrane. Millimolar
concentration of Cys dramatically increases ( congruent with 800%) the
activity of CopA and other P(IB)-type ATPases (Escherichia coli ZntA
and Arabidopsis thaliana HMA2). The high affinity of CopA for metal (
congruent with 1 microM) together with the low Cu(+)-Cys K(D)
(<10(-10)M) suggested a multifaceted interaction of Cys with CopA,
perhaps acting as a substitute for the Cu(+) chaperone protein present
in vivo. To explain the activation by the amino acid and further
understand the mechanism of metal delivery to transport ATPases, Cys
effects on the turnover and partial reactions of CopA were studied.
2-20 mM Cys accelerates enzyme turnover with little effect on CopA
affinity for Cu(+), suggesting a metal independent activation.
Furthermore, Cys activates the p-nitrophenyl phosphatase activity of
CopA, even though this activity is metal independent. Cys accelerates
enzyme phosphorylation and the forward dephosphorylation rates yielding
higher steady state phosphoenzyme levels. The faster dephosphorylation
would explain the higher enzyme turnover in the presence of Cys. The
amino acid has no significant effect on low affinity ATP K(m)
suggesting no changes in the E(1)<-->E(2) equilibrium.
Characterization of Cu(+) transport into sealed vesicles indicates that
Cys acts on the cytoplasmic side of the enzyme. However, the Cys
activation of truncated CopA lacking the N-terminal metal binding
domain (N-MBD) indicates that activation by Cys is independent of the
regulatory N-MBD. These results suggest that Cys is a non-essential
activator of CopA, interacting with the cytoplasmic side of the enzyme
while this is in an E1 form. Interestingly, these effects also point
out that Cu(+) can reach the cytoplasmic opening of the access path
into the transmembrane transport sites either as a free metal or a
Cu(+)-Cys complex.