Characterization of zinc-depleted alanyl-tRNA synthetase from Escherichia coli: role of zinc.
To evaluate the role of zinc in Escherichia coli alanyl-tRNA synthetase, hydrodynamic measurements and circular dichroism spectra were obtained for the zinc-depleted protein and compared with those of the native enzyme. At a protein concentration of 5 mg ml(-1), pH 7.5, the sedimentation coefficient (s(20,w)) was 6.3 S and was virtually independent of temperature between 10 and 37 degrees C, similar to the results reported for the native form. However, the s(20,w) now decreased significantly as the concentration increased, indicative of a possible change in conformation. The s(20,w) value did not appear to change as the pH was increased to 9.5. In standard buffer with 3.3 M added urea, a single peak with a s(20,w) of 3.6 S was obtained and with 6.6 M added urea, a peak with a s(20,w) of 2.7 S was seen. Added Gd-HCl (6 M) gave a single peak with s(20,w) of 2. 0 S. Like the native form, laser light scattering studies indicated some heterogeneity and a radius of 6.4 nm which was virtually independent of concentration and temperature in the range of 10-37 degrees C. At 25 degrees C, a diffusion coefficient (D(20,w)) of 3.3 x 10(-7) cm(2) s(-1) was obtained. The combination of s(0)(20,w) and D(20,w) yielded a molecular mass of approximately 179 kDa, which is slightly less than that reported for the native dimeric form (186 kDa). The intrinsic viscosity at 25 degrees C was extrapolated to 5. 3 ml g(-1), a value significantly higher than that reported for the native form, which increased with temperature. These results indicate some conformational and flexibility changes from the native to the zinc-depleted form, which may explain differences in activity. Furthermore, urea denaturation experiments demonstrate the role of zinc in stabilization of AlaRS structure.
Sood
S M
SM
Department of Biochemistry, Loma Linda University School of Medicine, Loma Linda, California, 92350, USA. ssood@som.llu.edu
Wu
M X
MX
Hill
K A
KA
Slattery
C W
CW
D000412:Alanine-tRNA Ligase / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D002942:Circular Dichroism; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D000412:Alanine-tRNA Ligase; D015032:Zinc