The Biochemical Characterization of Two Mutant Arginyl Transfer Ribonucleic Acid Synthetases from Escherichia coli K-12

Abstract

Abstract The arginyl transfer RNA synthetase from Escherichia coli K-12, wild-type, and two arginyl-tRNA synthetase mutant strains, MA 5003 and MA 5005, was purified approximately 200, 140, and 180 times, respectively. Various parameters of these synthetases were measured with the use of the esterification of arginine to transfer RNA as the assay. The mutant enzymes were found to have certain altered properties in common. Both mutant synthetases displayed an increased thermal sensitivity when compared to the wild-type enzyme. The pH optimum for all three enzymes was 8.0, but the activity of the mutant synthetases decreased more rapidly than the activity of the wild-type enzyme as the pH was lowered from 8.0 to 5.5. The Vmax of the mutant synthetases was lower than that of the wild-type enzyme when parameters as the Km values for arginine, ATP, tRNA, and dATP were measured. What differentiated the two mutant synthetases was the observation that the enzyme from strain MA 5003 displayed a 10- to 15-fold increase in Km for arginine, but less than a 2-fold increase in Km for ATP, whereas the arginyl-tRNA synthetase from strain MA 5005 had a 10-fold increase in Km for ATP, but less than a 2-fold increase in Km for arginine compared to the wild-type enzyme. It appears that the mutation in each mutant enzyme independently affects the Km for one substrate, arginine or ATP. When dATP was substituted for ATP, no difference in Km was observed between the wild-type enzyme and the mutant enzyme from strain MA 5005. Lastly the Km for tRNA was the same for all three enzymes.