HlyC, the internal protein acyltransferase that activates hemolysin toxin: roles of various conserved residues in enzymatic activity as probed by site-directed mutagenesis.
HlyC, the internal protein acyltransferase that activates hemolysin toxin: roles of various conserved residues in enzymatic activity as probed by site-directed mutagenesis.
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HlyC，激活溶血素毒素的内部蛋白酰基转移酶：通过定点诱变探测各种保守残基在酶活性中的作用。
DOI：
10.1021/bi9905617
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Ernst-Fonberg,ML
中科院分区：
文献类型：
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作者：
Trent,MS;Worsham,LM;Ernst-Fonberg,ML
溶血素是由致病性大肠杆菌产生的一种毒蛋白，是革兰氏阴性菌产生的同源毒素和毒素加工蛋白家族中的一员。HlyC是一种内部蛋白质酰基转移酶，将其从无毒的原溶血素转化为有毒的溶血素。酰基-酰基载体蛋白是必需的酰基供体。酰基转移酶反应通过酰基-ACP和HlyC之间的二元复合物形成反应性酰基-HlyC中间体而进行[Trent，M.美国，沃舍姆湖M.，和Ernst-Fonberg，M. L.（1998）Biochemistry 37，4644 - 4655]。该家族的同源酰基转移酶具有许多可能是催化重要的保守氨基酸残基。实验阐明了反应机理。酰基酶中间体的形成表明反应可能通过两个部分反应进行。通过使用分别亚克隆、纯化和表达的底物和酶显示第一部分反应的可逆性。确定了HlyC保守残基的单位点定向突变对反应进程（二元复合物形成，酰基酶形成和酶活性，包括动力学参数）的不同部分的影响。His 23的突变，活性所必需的唯一残基，形成正常的二元复合物，但不能形成酰基-HlyC。S20 A也是如此，它是一种活性大大受损的突变体。两个保守的酪氨酸分别突变为甘氨酸的结果大大受损的二元复合物和酰基-HlyC的形成，但这些残基突变为苯丙氨酸恢复野生型的行为。
Hemolysin, a toxic protein produced by pathogenicEscherichia coli, is one of a family of homologous toxins and toxin-processing proteins produced by Gram-negative bacteria. HlyC, an internal protein acyltransferase, converts it from nontoxic prohemolysin to toxic hemolysin. Acyl-acyl carrier protein is the essential acyl donor. The acyltransferase reaction progresses through formation of a binary complex between acyl-ACP and HlyC to a reactive acyl-HlyC intermediate [Trent, M. S., Worsham, L. M., and Ernst-Fonberg, M. L. (1998)Biochemistry 37, 4644−4655]. The homologous acyltransferases of the family have a number of conserved amino acid residues that may be catalytically important. Experiments to illuminate the reaction mechanism were done. The formation of an acyl-enzyme intermediate suggested that the reaction likely proceeded through two partial reactions. The reversibility of the first partial reaction was shown by using separately subcloned, purified, and expressed substrates and enzyme. The effects of single site-directed mutations of conserved residues of HlyC on different portions of reaction progress (binary complex formation, acyl-enzyme formation, and enzyme activity, including kinetic parameters) were determined. Mutations of His23, the only residue essential for activity, formed normal binary complexes but were unable to form acyl-HlyC. The same was seen with S20A, a mutant with greatly impaired activity. Mutation of two conserved tyrosines separately to glycines results in greatly impaired binary complex and acyl-HlyC formation, but mutation of those residues to phenylalanines restored behavior to wild-type.