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°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°C. At 25°C, a diffusion coefficient (D(20,w)) of 3.3 x 10^-7 cm² s^-1 was obtained. The combination of s(20,w)/⁰ 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°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.