POSTTRANSLATIONAL SYNTHESIS OF HYPUSINE IN EIF-5A--DEOXYHYPUSINE SYNYTHASE

一种不常见的氨基酸，羟腐胺赖氨酸，只存在于一种细胞中， 真核生物翻译起始因子5A（eIF-5A）是一种蛋白质， 与真核细胞增殖密切相关。羟腐 生物合成通过两个连续的翻译后 修饰反应：1）脱氧羟腐胺赖氨酸合成和2）脱氧羟腐胺赖氨酸 羟基化 脱氧羟腐胺赖氨酸合酶催化 多胺亚精胺的丁胺部分与特定的赖氨酸残基连接 在eIF-5A前体蛋白中形成脱氧羟腐胺赖氨酸。我们净化了 这种酶来自大鼠睾丸，在酵母中鉴定了其基因 酿酒酵母中，并克隆了人的cDNA。失活 酵母中的脱氧羟腐胺赖氨酸合酶基因导致细胞活力丧失， 表明羟腐胺赖氨酸修饰对于体内eIF-5A是至关重要的， 活性和细胞增殖。我们已经描述了物理和 酶的催化性质和反应机理。我们有 鉴定了人酶的活性位点残基（Lys 329）， 参与酶中间体的形成，因此对于 催化作用X射线晶体学（与D. I.廖 和D. R. Davies）的人脱氧羟腐胺赖氨酸合酶与NAD的复合物 揭示了它的四聚体结构和NAD结合位点。分子 亚精胺结合位点的建模应该有助于开发特异性的 脱氧羟腐胺赖氨酸合酶的抑制剂，其可用作抗- 增殖剂。

An unusual amino acid, hypusine, which occurs in only one cellular protein, eukaryotic translation initiation factor 5A (eIF-5A), is intimately involved in eukaryotic cell proliferation. Hypusine biosynthesis occurs by way of two sequential post-translational modification reactions: 1) deoxyhypusine synthesis and 2) deoxyhypusine hydroxylation. Deoxyhypusine synthase catalyzes the transfer of the butylamine moiety of the polyamine spermidine to a specific lysine residue in the eIF-5A precursor protein to form deoxyhypusine. We have purified this enzyme from rat testis, identified its gene in the yeast Saccharomyces cerevisiae and cloned the human cDNA. Inactivation of the deoxyhypusine synthase gene in yeast causes loss of cell viability, indicating that the hypusine modification is vital for in vivo eIF-5A activity and cell proliferation. We have characterized the physical and catalytic properties and the reaction mechanism of the enzyme. We have identified an active site residue (Lys329) of the human enzyme that is involved in enzyme-intermediate formation, and thereby is critical for catalysis. X-ray crystallography (in collaboration with Drs. D.-I. Liao and D. R. Davies) of human deoxyhypusine synthase in a complex with NAD has revealed its tetrameric structure and the NAD binding site. Molecular modeling of the spermidine binding site should aid development of specific inhibitors of deoxyhypusine synthase that may be useful as anti- proliferative agents.