Further characterization of Escherichia coli alanyl-tRNA synthetase

Selected physical and thermodynamic parameters for Escherichia coli alanyl-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^-1 cm^-1, and the partial specific volume (v̄) 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^-1 and the degree of hydration (δ₁) estimated to be 0.67 g(H2O) g(AlaRS)^-1. Laser light-scattering studies indicated some heterogeneity; a radius of 6.3 nm was calculated for the major fraction with a diffusion coefficient (D(20,w)) of 3.89 x 10^-7 cm² s^-1. In 50 mM Depes, pH 7.5, 20 mM KCl, 2 mM 2-mercaptoethanol and at a protein concentration of 4.2 mg ml^-1 the sedimentation coefficient (s(20,w)) was 6.36 S; this value increased slightly when the protein concentration was decreased. The combination of s(20,w) and D(20,w) under these conditions yielded a molecular weight of approximately 186,000 Da, corresponding to a dimer. The s(20,w) was 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.