J. Biol. Chem., Vol. 261, Issue 16, 7264-7269, Jun, 1986
Characterization of rhodanese-tetracyanonickelate. An active site complex that slows sulfur-free rhodanese conversion to inert conformers
SF Chow and P Horowitz
The structure of the rhodanese-tetracyanonickelate (E X Ni(CN)2-4) complex
has been characterized here in spectral and physical studies using urea as
a structural perturbant. UV difference absorption, sedimentation velocity
ultracentrifugation, fluorescence, and circular dichroism data show no
significant conformational differences between sulfur-free rhodanese (E)
and the E X Ni(CN)2-4 complex. The urea- induced enzyme structural
transition curves were noncoincident when different structural parameters
were monitored. For E, the urea concentrations giving half-maximal change
(Cm) were: Cm = 3.0 M for activity measurement; Cm = 2.8 M for protein
intrinsic fluorescence intensity; Cm = 4.3 M for ellipticity at 220 nm; and
Cm = 3.3 M for wavelength of fluorescence emission maximum. For the E X
Ni(CN)2-4 complex, Cm was shifted to a higher urea concentration relative
to that found for E when activity (Cm = 3.6 M) and native protein
fluorescence (Cm = 3.6 M) were the measured parameters but not when the
wavelength of the emission maximum and ellipticity were monitored.
Furthermore, urea-induced rhodanese structural changes were time-dependent
and Ni(CN)2-4 binding on E slowed enzyme inactivation that is associated
with structural relaxations. These findings, that Ni(CN)2-4 affects
structural relaxations in rhodanese, are of particular interest in light of
the recent suggestion that the E X Ni(CN)2-4 complex mimics a normally
inaccessible intermediate in catalysis.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?