Further Characterization ofEscherichia coliAlanyl–tRNA Synthetase

Selected physical and thermodynamic parameters forEscherichia colialanyl–tRNA synthetase (AlaRS) have been determined primarily to assess the quaternary structure of this enzyme. The extinction coefficient (ϵ) at 280 nm was determined experimentally to be 0.71 ml mg−1cm−1, and the partial specific volume (ν) was calculated from the amino acid composition to be 0.73 ml g−1. From viscosity experiments the intrinsic viscosity ([η]) of AlaRS was extrapolated to be 3.4 ml g−1and the degree of hydration (δ1) estimated to be 0.67 gH2Og−1AlaRS. Laser light-scattering studies indicated some heterogeneity; a radius of 6.3 nm was calculated for the major fraction with a diffusion coefficient (D20,w) of 3.89 × 10−7cm2s−1. In 50 mMHepes, pH 7.5, 20 mMKCl, 2 mM2-mercaptoethanol and at a protein concentration of 4.2 mg ml−1the sedimentation coefficient (s20,w) was 6.36 S; this value increased slightly when the protein concentration was decreased. The combination ofs20,wandD20,wunder these conditions yielded a molecular weight of approximately 186,000 Da, corresponding to a dimer. Thes20,wwas virtually independent of temperature in the range of 10–37°C, while an Arrhenius plot of aminoacylation activity was biphasic. The isoelectric point was determined experimentally to be 4.9. Sedimentation equilibrium data were best fit to a decamer association complex in which dimeric AlaRS is the predominant species at 25°C.