DEOXYHYPUSINE SYNTHASE--PURIFICATION, CHARACTERIZATION, AND CDNA CLONING

一种不寻常的氨基酸，苏氨酸，只存在于一种细胞中 真核细胞翻译分离因子5A(eIF-5A)，是 与细胞增殖密切相关。Hypusine的生物合成发生 通过两个顺序的翻译后修饰反应：i) 脱氧亚硫氨酸合成酶合成脱氧亚硫氨酸和II)脱氧亚硫氨酸 脱氧亚精氨酸羟基酶的羟化作用。 我们已经将第一种酶，脱氧亚硫氨酸合成酶提纯为均一酶。 在大鼠睾丸中进行了大约10万倍的浓缩后。纯化的酶 对其底物表现出极窄的专一性， 亚精胺、NAD和eIF-5A前体蛋白及其催化 在完全反应混合物中合成脱氧亚精氨酸。在缺席时 然而，对于底物蛋白质，它会进行部分反应，即 亚精胺的NAD依赖的裂解。这种酶以四聚体的形式存在 42 kDa亚基，等电点为4.75。 利用大鼠睾丸酶的部分氨基酸序列，我们 酿酒酵母第八号染色体YHRO68W的鉴定 脱氧亚精氨酸合酶基因。我们还克隆了编码人类DNA的cDNA HeLa细胞全长脱氧亚硫氨酸合成酶的免疫筛选 C DNA文库。人脱氧亚硫氨酸合成酶过表达后 YHRO68W的酵母基因在大肠杆菌中的表达，我们纯化了重组 并对其物理和酶学性质进行了表征。 删除、插入和定点突变研究正在进行中 深入了解活动站点的结构和结构功能 这种重要的酶之间的关系。

An unusual amino acid, hypusine, which occurs in only one cellular protein, eukaryotic translation isolation factor 5A (eIF-5A), is intimately involved in cell proliferation. Hypusine biosynthesis occurs by way of two sequential post-translational modification reactions: i) deoxyhypusine synthesis by deoxyhypusine synthase and ii) deoxyhypusine hydroxylation by deoxyhypusine hydroxylase. We have purified the first enzyme, deoxyhypusine synthase, to homogeneity after about 100,000-fold enrichment from rat testis. The purified enzyme displays a remarkably narrow specificity toward its substrates, spermidine, NAD, and the eIF-5A precursor protein and catalyzes deoxyhypusine synthesis in the complete reaction mixture. In the absence of the substrate protein, however, it carries out a partial reaction, the NAD-dependent cleavage of spermidine. The enzyme exists as a tetramer of 42 kDa subunits, with a pI of 4.75. Using partial amino acid sequences from the rat testis enzyme, we were able to identify YHRO68W of Saccharomyces cerevisiae chromosome VIII as a gene for deoxyhypusine synthase. We have also cloned human cDNAs encoding a full-length deoxyhypusine synthase by immunoscreening of a HeLa cell cDNA library. After overexpression of the human deoxyhypusine synthase cDNA or the yeast cDNA of YHRO68W in E. coli, we purified the recombinant enzymes and characterized their physical and enzymatic properties. Deletion, insertion and site-directed mutagenesis studies are underway to gain insights into the active site structure and the structure-function relationship of this important enzyme.