Mikulka T W, Stieglitz B I, Calvo J M
J Bacteriol. 1972 Feb;109(2):584-93. doi: 10.1128/jb.109.2.584-593.1972.
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.
从鼠伤寒沙门氏菌提取物中纯化出100倍的亮氨酰 - 转移核糖核酸（tRNA）合成酶。部分纯化的酶具有以下米氏常数（K(m)）值：亮氨酸，1.1×10⁻⁵m；三磷酸腺苷，6.5×10⁻⁴m；tRNA(I)（Leu），4.1×10⁻⁸m；tRNA(II)（Leu），4.3×10⁻⁸m；tRNA(III)（Leu），5.3×10⁻⁸m；以及tRNA(IV)（Leu），2.9×10⁻⁸m。通过反相柱和苯甲酰化二乙氨基乙基纤维素色谱法从沙门氏菌总tRNA中分离出tRNA(Leu)组分。该酶的最适pH为8.5，活化能为每摩尔10400卡，在49.5℃时以0.064 min⁻¹的一级速率常数呈指数失活。菌株CV356（leuS3 leuABCD702 ara - 9 gal - 205）作为对dl - 4 - 氮杂亮氨酸有抗性且能在27℃生长但不能在37℃生长的突变体被分离出来。当在27℃或37℃进行测定时，菌株CV356的提取物没有亮氨酰 - tRNA合成酶活性（充电测定）。温度敏感性和酶缺陷是由指定亮氨酰 - tRNA合成酶的结构基因位点突变引起的。菌株CV356的原养型衍生物（CV357）在其倍增时间接近正常的温度下生长时会分泌支链氨基酸并且具有高途径特异性酶水平。在生长受限温度下，氨基酸分泌和酶水平都会进一步升高。菌株CV357的特性表明鼠伤寒沙门氏菌中只有一种亮氨酰 - tRNA合成酶。
