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 (epsilon) at 280 nm was determined experimentally to be 0.71 ml mg(-1) cm(-1), and the partial specific volume (<(nu)over bar>) was calculated from the amino acid composition to be 0.73 ml g(-1). From viscosity experiments the intrinsic viscosity ([eta]) of AlaRS was extrapolated to be 3.4 ml g(-1) and the degree of hydration (delta(1)) 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,D-w) of 3.89 x 10(-7) cm(2) s(-1). In 50 mM Hepes, 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,D-w under these conditions yielded a molecular weight of approximately 186,000 La, corresponding to a dimer. The s(20,w) was virtually independent of temperature in the range of 10-37 degrees 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 degrees C. (C) 1996 Academic Press, Inc.