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

Abstract

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 molecu- lar 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 as- sayed in the temperature range SO-70°C under condi- tions 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 ATR- pyrophosphate exchange reactions. This inhibition could not be related to the melting of the tRNA sub- strates 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 grad- ual increase with temperature. The inhibition observed could be explained by assuming a temperature-induced conformational change of the enzymes affecting pri- marily the binding sites for ATP and amino acid. The amino acid:tRNA ligases play a central role in the translation of geneic information into amino acid sequences in proteins. The mechanism by which they select their tRNA substrates is also an interesting example of a specific interac- tion between an enzyme and a nucleic acid and has conse- quently received much attention. In the final analysis our understanding of this process must be based on a detailed knowledge of the structure of both enzyme and tRNA. Several ligases’ have been crystallixed by now (l-5), and for one of them, the tyrosine enzyme from Bacillus stearothermophilus, a low resolution structure has been obtained (4), and a struc- ture at high resolution can be expected in the near future. A low resolution structure for an enxymatically active major