Lee Y B, Joe Y A, Wolff E C, Dimitriadis E K, Park M H
Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892-4340, USA.
Biochem J. 1999 May 15;340 ( Pt 1)(Pt 1):273-81.
Deoxyhypusine synthase catalyses the first step in the post-translational synthesis of hypusine [Nepsilon-(4-amino-2-hydroxybutyl) lysine] in a single cellular protein, the precursor of eukaryotic initiation factor 5A (eIF5A). Deoxyhypusine synthase exists as a tetramer with four potential active sites. The formation of a stable complex between human deoxyhypusine synthase and its protein substrate, human recombinant eIF5A precursor (ec-eIF5A), was examined by affinity chromatography using polyhistidine-tagged (His.Tag) ec-eIF5A, by a gel mobility-shift method, and by analytical ultracentrifugation. Deoxyhypusine synthase was selectively retained by His.Tag-ec-eIF5A immobilized on a resin. The complex of deoxyhypusine synthase and ec-eIF5A was separated from the free enzyme and protein substrate by electrophoresis under non-denaturing conditions. The stoichiometry of the two components in the complex was estimated to be 1 deoxyhypusine synthase tetramer to 1 ec-eIF5A monomer by N-terminal amino acid sequencing of the complex. Equilibrium ultracentrifugation data further supported this 1:1 ratio and indicated a very strong interaction of the enzyme with ec-eIF5A (Kd</=0.5 nM). Formation of the complex was not dependent on NAD+ or spermidine and occurred at pH7.0-9.2. An enzyme-product complex, as well as the deoxyhypusine-containing product (modified ec-eIF5A), was also detected at pH7.0-9.2 in a complete reaction mixture containing 1 mM spermidine.
脱氧hypusine合酶催化单细胞蛋白——真核起始因子5A（eIF5A）前体中hypusine（Nε-（4-氨基-2-羟丁基）赖氨酸）翻译后合成的第一步。脱氧hypusine合酶以具有四个潜在活性位点的四聚体形式存在。通过使用多组氨酸标签（His.Tag）的ec-eIF5A进行亲和色谱、凝胶迁移率变动法和分析超速离心，研究了人脱氧hypusine合酶与其蛋白底物人重组eIF5A前体（ec-eIF5A）之间稳定复合物的形成。固定在树脂上的His.Tag-ec-eIF5A选择性保留了脱氧hypusine合酶。在非变性条件下通过电泳将脱氧hypusine合酶与ec-eIF5A的复合物与游离酶和蛋白底物分离。通过复合物的N端氨基酸测序估计复合物中两种成分的化学计量比为1个脱氧hypusine合酶四聚体比1个ec-eIF5A单体。平衡超速离心数据进一步支持了这种1:1的比例，并表明该酶与ec-eIF5A有非常强的相互作用（Kd≤0.5 nM）。复合物的形成不依赖于NAD⁺或亚精胺，且在pH7.0 - 9.2时发生。在含有1 mM亚精胺的完整反应混合物中，在pH7.0 - 9.2时也检测到了酶-产物复合物以及含脱氧hypusine的产物（修饰的ec-eIF5A）。
