Leucyl-transfer ribonucleic acid synthetase from a wild-type and temperature-sensitive mutant of Salmonella typhimurium.
Leucyl-transfer ribonucleic acid (tRNA) synthetase was purified 100-fold from extracts of Salmonella typhimurium. The partially purified enzyme had the following K(m) values: leucine, 1.1 x 10(-5)m; adenosine triphosphate, 6.5 x 10(-4)m; tRNA(I) (Leu), 4.1 x 10(-8)m; tRNA(II) (Leu), 4.3 x 10(-8)m; tRNA(III) (Leu), 5.3 x 10(-8)m; and tRNA(IV) (Leu), 2.9 x 10(-8)m. The tRNA(Leu) fractions were isolated from Salmonella bulk tRNA by chromatography on reversed-phase columns and benzoylated diethylaminoethyl cellulose. The enzyme had a pH optimum of 8.5 and an activation energy of 10,400 cal per mole, and was inactivated exponentially at 49.5 C with a first-order rate constant of 0.064 min(-1). Strain CV356 (leuS3 leuABCD702 ara-9 gal-205) was isolated as a mutant resistant to dl-4-azaleucine and able to grow at 27 C but not at 37 C. Extracts of strain CV356 had no leucyl-tRNA synthetase activity (charging assay) when assayed at 27 or 37 C. Temperature sensitivity and enzyme deficiency were caused by mutation in the structural gene locus specifying leucyl-tRNA synthetase. A prototrophic derivative of strain CV356 (CV357) excreted branched-chain amino acids and had high pathway-specific enzyme levels when grown at temperatures where its doubling time was near normal. At growth-restricting temperatures, both amino acid excretion and enzyme levels were further elevated. The properties of strain CV357 indicate that there is only a single leucyl-tRNA synthetase in S. typhimurium.
D000255:Adenosine Triphosphate / Q000032:analysis; D000332:Aerobiosis; D000596:Amino Acids / Q000032:analysis; D000604:Amino Acyl-tRNA Synthetases / Q000302:isolation & purification* / Q000378:metabolism; D001322:Autoanalysis; D002247:Carbon Isotopes; D002474:Cell-Free System; D002848:Chromatography, DEAE-Cellulose; D004352:Drug Resistance, Microbial; D004794:Enzyme Repression; D005796:Genes; D006863:Hydrogen-Ion Concentration; D007930:Leucine / Q000032:analysis; D009154:Mutation; D012343:RNA, Transfer / Q000032:analysis; D012486:Salmonella typhimurium / Q000032:analysis / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development / Q000302:isolation & purification; D013696:Temperature; D014161:Transduction, Genetic
D000596:Amino Acids; D002247:Carbon Isotopes; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; D007930:Leucine