IQUIFIB   02644
INSTITUTO DE QUIMICA Y FISICOQUIMICA BIOLOGICAS "PROF. ALEJANDRO C. PALADINI"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Pre-steady kinetic of ATP hydrolysis by dengue virus NS3 helicase in single turnover conditions
Autor/es:
RODOLFO M GONZÁLEZ LEBRERO; LG GEBHARD; AV GAMARNIK; SB. KAUFMAN
Lugar:
Salta
Reunión:
Congreso; XXXIX Annual Meeting of the Argentinean Biophysical Society (SAB) and 3rd Latin American Protein Society Meeting; 2010
Institución organizadora:
Sociedad Argentina de Biofísica
Resumen:
The NS3
dengue virus helicase is a molecular motor protein that unwinds duplex RNA
during the replication of the viral genome. The binding and hydrolysis of
adenosine triphosphate (ATP) are essential for helicase function. However, the
reaction mechanism that explains the coupling between nucleotide hydrolysis and
duplex RNA unwinding is still unknown. Thus, we propose to study the kinetic
mechanism of nucleotide hydrolysis during the catalytic cycle of NS3. According
to this, we performed pre-steady state experiments of ATP hydrolysis by
following orthophosphate(Pi) release in single turnover conditions. The
time courses of Pi release were obtained at different concentrations of ATP and
NS3 but, in all cases, with excess of enzyme on substrate and pseudo-first
order conditions. When the enzyme concentration was 1.022 µM and [ATP] = 0.2,
0.051 and 0.00019 µM the time courses of Pi release were monoexponential curves
with apparent rate contants: 0.048, 0.048 and 0.053 s-1
respectively. When the enzyme concentration was decreased to 0.050 and 0.014 µM
with [ATP] = 0.00019 and 0.00016 µM also monoexponential curves were obtained
with apparent rates constants: 0.0054 and 0.0019 s-1 respectively.
These results suggests that, in the present experimental conditions the rate
limiting step of ATP hydrolysis is the formation of collisional complex NS3-ATP
with an apparent second order rate constant of 0.0466 µM-1 s‑1.
NS3 protein was cloned
from the dengue virus 2 16681 infectious clone. The recombinant protein was
purified by histidine‑tag affinity chromatography. All time courses were
carried-out at 25 °C
in media containing: 100 mM
KCl; 25 mM
MOPS/K (pH=6.5); 2 mM
MgCl2; 0.5 mM
EDTA; 3% glycerol.
With grant B412 UBACyT.