Leukotriene A4 hydrolase: determination of the three zinc-binding ligands by site-directed mutagenesis and zinc analysis.
Leukotriene A4 hydrolase: determination of the three zinc-binding ligands by site-directed mutagenesis and zinc analysis.
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白三烯 A4 水解酶：通过定点诱变和锌分析测定三种锌结合配体。
DOI：
10.1073/pnas.88.17.7620
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发表时间：
1991
影响因子：
11.1
通讯作者：
B. Samuelsson
中科院分区：
文献类型：
--
作者：
J. F. Medina;A. Wetterholm;O. Rådmark;R. Shapiro;J. Haeggström;B. Vallée;B. Samuelsson
利用cDNA定点诱变技术，制备了3个重组小鼠白三烯A4 （LTA4）水解酶（3.3.2.6）突变体。对应于His-295、His-299和Glu-318的密码子分别被编码酪氨酸、酪氨酸和谷氨酰胺的密码子所取代。在大肠杆菌中表达突变的cdna，并纯化了三个突变蛋白，具有明显的同质性。通过原子吸收光谱测定，这些突变体都不含大量的锌，而且它们实际上都缺乏LTA4水解酶和肽酶的活性。然而，在免疫印迹分析中，突变蛋白可以通过其与人LTA4水解酶抗血清的免疫反应性得到阳性鉴定。对人LTA4水解酶cDNA也进行了定点诱变。编码His-295、His-299和Glu-318的密码子分别被编码酪氨酸、亮氨酸和丙氨酸的密码子取代，这三个突变体在大肠杆菌中表达。在这些表达中产生的总可溶性蛋白的LTA4水解酶活性低于含有非突变cDNA的细菌的10%。与先前的预测一致，我们的实验数据表明，His-295、His-299和Glu-318构成了LTA4水解酶中固有锌原子的三个配体。此外，纯化的突变小鼠蛋白中酶活性和锌含量的综合损失强调了锌原子在催化中的关键作用，而突变和未突变小鼠LTA4水解酶蛋白的几乎相同的色谱行为表明金属对维持酶三级结构的重要性有限。
Three mutants of recombinant mouse leukotriene A4 (LTA4) hydrolase (3.3.2.6) were produced by site-directed mutagenesis on cDNA. The codons corresponding to His-295, His-299, or Glu-318 were replaced by codons encoding tyrosine, tyrosine, and glutamine, respectively. The mutated cDNAs were expressed in Escherichia coli, and the three mutated proteins were purified to apparent homogeneity. None of these mutants contained significant amounts of zinc, as determined by atomic absorption spectrometry, and all of them were practically devoid of both LTA4 hydrolase and peptidase enzyme activities. Nevertheless, the mutated proteins could be positively identified by their immunoreactivities with an antiserum for human LTA4 hydrolase in immunoblot analysis. Site-directed mutagenesis was also carried out on human LTA4 hydrolase cDNA. Codons encoding His-295, His-299, and Glu-318 were replaced by ones encoding tyrosine, leucine, and alanine, respectively, and the three mutants were expressed in E. coli. The LTA4 hydrolase activities of the total soluble proteins produced in these expressions were less than 10% of that obtained for bacteria harboring nonmutated cDNA. In agreement with earlier predictions, our experimental data demonstrate that His-295, His-299, and Glu-318 constitute the three ligands of the intrinsic zinc atom in LTA4 hydrolase. Additionally, the combined loss of enzyme activities and zinc content in the purified mutated mouse proteins, emphasizes the critical role of the zinc atom for catalysis, whereas the virtually identical chromatographic behaviors of the mutated and nonmutated mouse LTA4 hydrolase proteins suggest that the metal is of limited importance for the maintenance of the enzyme tertiary structure.