Purification and some properties of asparagine, lysine, serine, and valine:tRNA ligases from Bacillus stearothermophilus.

A procedure for the purification of the asparagine, lysine, serine, and valine:tRNA ligases from the same batch of Bacillus stearothemophilus is described.The purified enzymes were apparently homogeneous by polyacrylamide gel electrophoresis.When the molecular weights of the asparagine, lysine, and serine:tRNA ligases, determined under nondenaturing conditions by equilibrium centrifugation and velocity sedimentation, were compared with the molecular weights obtained under denaturing conditions, these enzymes appeared to contain two subunits of equal molecular weight per molecule of active enzyme.The catalytic properties at high temperatures of these enzymes and of the valine:tRNA ligase from the same organism have also been investigated.When assayed in the temperature range SO-70°C under conditions when there was no measurable loss of catalytic activity, all enzymes showed a marked deviation from linearity in the Arrhenius plots indicating a reversible inhibition both of the aminoacylation and the ATRpyrophosphate exchange reactions.This inhibition could not be related to the melting of the tRNA substrates as measured by hyperchromicity.On the other hand, the K,,,values for ATP and amino acid increased sharply in this temperature range in contrast to the K,,, values for tRNA which showed only the expected gradual increase with temperature.The inhibition observed could be explained by assuming a temperature-induced conformational change of the enzymes affecting primarily the binding sites for ATP and amino acid.