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Leucyl-Transfer Ribonucleic Acid Synthetase from a Wild-Type and Temperature-Sensitive Mutant of Salmonella typhimurium¹

^a Section of Biochemistry and Molecular Biology, Division of Biological Sciences, Cornell University, Ithaca, New York 14850

ABSTRACT

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^–5M; adenosine triphosphate, 6.5 x 10^–4M; tRNA_I^Leu, 4.1 x 10^–8M; tRNA_II^Leu, 4.3 x 10^–8M; tRNA_III^Leu, 5.3 x 10^–8M; and tRNA_IV^Leu, 2.9 x 10^–8M. 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.

² Present address: National Institute of Arthritis and Metabolic Disease, Bethesda, Md. 20014.

³ Present address: Department of Applied Microbiology, Hebrew University-Hadassah Medical School, Jerusalem, Israel.

¹ This report is part of a dissertation presented by the senior author in partial fulfillment of the requirements for the Ph.D. degree from Cornell University.