Abstract
Alanyl- and leucyl-tRNA synthetases from baker's yeast were purified to homogeneity in the presence of the protease inhibitor phenylmethylsulfonyl fluoride. Both consist of single polypeptide chains of 118 000 and 125 000 daltons, respectively, as determined by polyacrylamide gel electrophoresis under denaturing conditions. The monomeric structure of leucyl-tRNA synthetase differs from the dimeric one obtained previously in the absence of protease inhibitors. This illustrates the sensitivity of the synthetases to proteolytic actions and indicates that native structures can only be obtained under optimal protecting conditions. Alanyl- and leucyl-tRNA synthetases differ with respect to pH optimum (6.5 and 8.5, respectively), Michaelis constant for amino acid (1 mM and 0.03, respectively) and in the rate-limiting step for the tRNA aminoacylation reaction. Whereas the catalytic step itself was rate-limiting for alanyl-tRNA synthetase, a step occurring after this was rate-limiting for leucyl-tRNA synthetase.