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) 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 gH2O g(-1)(AlaRS). 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 x 10(-7) cm2 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 (S20,W) was 6.36 S; this value increased slightly when the protein concentration was decreased. The combination of S20,W and D20,W under these conditions yielded a molecular weight of approximately 186,000 Da, corresponding to a dimer. The S20,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.
Sood
S M
SM
Department of Biochemistry, Loma Linda University School of Medicine, California 92350-0001, USA.
Slattery
C W
CW
Filley
S J
SJ
Wu
M X
MX
Hill
K A
KA
D000412:Alanine-tRNA Ligase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D007834:Lasers; D008027:Light; D046911:Macromolecular Substances; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012542:Scattering, Radiation; D013816:Thermodynamics; D014783:Viscosity
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D000412:Alanine-tRNA Ligase