THE PURIFICATION AND STUDIES OF KINETICS OF ESCHERICHIA-COLI LEUCYL-TRANSFER-RNA SYNTHETASE MUTANT (LEURS67R)

The gene coding for leucyl-tRNA synthetase (LeuRS) has been cloned from E. coli K-12. There is a substitute of G for A at the position of base 200 in the gene compared with the gene from E. coli K-12 1100. Because His67 of this enzyme is changed to Arg. the enzyme was designated LeuRS67R. We have got over expression of this enzyme from E. coli TG1 transformant strain which harbors the recombination plasmid pUC19 with the gene. In the crude extract of the transformant strain LeuRS67R was overproduced. 20 times more than that of the wild-type TG1 strain. The purified enzyme showing one band on SDS-PAGE has been obtained by three steps of column chromatography. The specific activity of the preparation was 1786 units/mg. For the aminoacylation reaction the K values at pH7.4, 37 degrees C, for Leu and ATP were determined to be 0.027 mM and 0.47 mM, respectively, k(cat) was 3.5 similar to 5.1s(-1). The K-m values for ATP-PPi exchange reaction were found to be 0.03 mM and 0.75 mM for leucine and ATP, respectively, k(cat) was 140 similar to 155(s-1). The result has shown there is a little difference in the kinetic constants between LeuRS67R and LeURS previously obtained from E. coli K-12. This mutant at 67 amino acid residue map not be involved in the active site.