THE POST-TRANSLATIONAL SYNTHESIS OF HYPUSINE IN EIF5A: DEOXYHYPUSINE SYNTHASE

在以前的研究中，我们已经确定eIF 5A作为唯一的细胞蛋白，含有一种不寻常的氨基酸，羟腐胺赖氨酸[Nepaline-（4-氨基-2-羟丁基）赖氨酸]，并建立了羟腐胺赖氨酸生物合成发生两个连续的酶促反应，脱氧羟腐胺赖氨酸合成和脱氧羟腐胺赖氨酸羟基化。其在eIF 5A前体中在单个赖氨酸残基处的合成代表了迄今为止已知的最特异性的翻译后修饰。羟腐胺赖氨酸对于eIF-5A的活性和真核细胞增殖是必需的。羟腐胺赖氨酸生物合成的抑制剂导致细胞增殖停滞。脱氧羟腐胺赖氨酸合酶催化多胺亚精胺的丁胺部分转移至eIF-5A前体蛋白中的特定赖氨酸残基以形成脱氧羟腐胺赖氨酸。我们从大鼠睾丸中纯化了该酶，在酿酒酵母中鉴定了其基因，并克隆了人cDNA。酵母中脱氧羟腐胺赖氨酸合酶基因的失活导致细胞活力的丧失，提供了羟腐胺赖氨酸修饰对酵母细胞生长至关重要的直接证据。对该酶的物理性质、催化性质和反应机理进行了表征。我们已经表明脱氧羟腐胺赖氨酸合成通过四个步骤发生：i）亚精胺的NAD依赖性脱氢，ii）丁胺部分从脱氢亚精胺转移到酶的特定赖氨酸以形成酶-亚胺中间体，iii）丁胺部分从酶中间体转移到eIF 5A前体，iv）eIF 5A亚胺中间体还原为含脱氧羟腐胺赖氨酸的形式。参与酶中间体形成的活性位点残基已被鉴定为人酶的Lys-329和酵母酶的Lys-350;因此，脱氧羟腐胺赖氨酸合成的机制似乎在真核生物中是保守的。与NAD复合的人脱氧羟腐胺赖氨酸合酶的X射线晶体结构（与Drs.D. -J. I. Liao和D. R. Davies，NIDDK）揭示了NAD结合位点和推测亚精胺结合的活性位点口袋。通过定点诱变证实了参与NAD、亚精胺结合的许多氨基酸以及那些对催化至关重要的氨基酸的作用。亚精胺结合位点的分子模拟应该有助于开发可能用作抗增殖剂的脱氧羟腐胺赖氨酸合酶的特异性抑制剂。- 翻译后修饰，eIF 5A，脱氧羟腐胺赖氨酸合酶，羟腐胺赖氨酸，多胺，细胞增殖

In previous studies we have identified eIF5A as the only cellular protein that contains an unusual amino acid, hypusine [Nepsilon-(4-amino-2-hydroxybutyl)lysine], and have established that hypusine biosynthesis occurs by two sequential enzymatic reactions, deoxyhypusine synthesis and deoxyhypusine hydroxylation. Its synthesis in the eIF5A precursor at a single lysine residue represents the most specific post-translational modification known to date. Hypusine is essential for the activity of eIF-5A and for eukaryotic cell proliferation. Inhibitors of hypusine biosynthesis cause arrest in cell proliferation. 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 providing direct evidence that the hypusine modification is vital for growth of yeast cells. We have characterized the physical and catalytic properties and the reaction mechanism of the enzyme. We have shown that deoxyhypusine synthesis occurs by way of four steps: i) NAD- dependent dehydrogenation of spermidine, ii) transfer of the butyl amine moiety from dehydrospermidine to a specific lysine of the enzyme to form an enzyme-imine intermediate, iii) transfer of the butylamine moiety from the enzyme intermediate to the eIF5A precursor, iv) reduction of the eIF5A imine intermediate to the deoxyhypusine-containing form. The active site residue that is involved in enzyme-intermediate formation has been identified asLys-329 for the human enzyme and Lys-350 for the yeast enzyme; thus, the mechanism of deoxyhypusine synthesis appears to be conserved in eukaryotes. The X-ray crystal structure of human deoxyhypusine synthase in a complex with NAD (determined in collaboration with Drs. D.-I. Liao and D. R. Davies, NIDDK) reveals NAD binding sites and an active site pocket where spermidine is presumed to bind. The role of a number of amino acids involved in the binding of NAD, of spermidine, and those critical for the catalysis, was confirmed by site-directed mutagenesis. Molecular modeling of the spermidine binding site should aid development of specific inhibitors of deoxyhypusine synthase that may be useful as anti-proliferative agents. - post-translational modification, eIF5A, deoxyhypusine synthase, hypusine, polyamines, cell proliferation