Detection of ligand-induced conformational changes in phenylalanyl-tRNA synthetase of Escherichia coli K10 by laser light scattering

Abstract

The diffusion constant of phenylalanyl-tRNA synthetase (EC 6.1.1.20) was measured by laser light scattering under conditions of complex formation with Mg2+, L-phenylalanine, MgATP, tRNAPhe, modified tRNAPhe, tRNAPhe (yeast) and noncognate tRNA. The diffusion constant (pH 7.5, 20.degree. C) of the free enzyme is (2.85 .+-. 0.005) .times. 10-7 cm2 s-1, of the enzyme.cntdot.Mg2+ complex (2.40 .+-. 0.05) .times. 10-7 cm2 s-1 and of the enzyme.cntdot.Mg2+.cntdot.tRNAPhe complex (2.95 .+-. 0.06) .times. 10-7 cm2 s-1. The effect of tRNAPhe is only seen with the enzyme is saturated with Mg2+. The smaller substrates exhibit no effect besides a small increase of the value of the diffusion constant under conditions where the enzyme-phenylalanyladenylate is synthesized. Of the noncognate tRNATyr and tRNAIle, the latter is able to associate with the enzyme, causing the value of the diffusion constant to increase. tRNAPhe (yeast) and .**GRAPHIC**. (photo-cross-linked tRNAPhe) exhibit similar effects. The observed variation of the diffusion constant is attributed to conformational changes of the enzyme. The opposite effects of Mg2+ and tRNAPhe are interpreted as an expansion and recontraction, respectively, of the enzyme molecule. In several cases, the effects were used to follow a titration of the enzyme with a ligand. Dissociation constants were calculated from the resulting titration curves, yielding values which are in agreement with those obtained by other techniques. Of the 2 possible binding sites for each Mg2+ and tRNAPhe, the diffusion constant reflects occupation of only a single class of sites.