Complex formation between deoxyhypusine synthase and its protein substrate, the eukaryotic translation initiation factor 5A (eIF5A) precursor

• Published in: Biochemical journal 1999-05, Vol.340 ( Pt 1) (1), p.273-281
• Main Authors: Lee, Y B, Joe, Y A, Wolff, E C, Dimitriadis, E K, Park, M H
• Format: Article
• Language: English
• Subjects: Cell Extracts; Chromatography, Affinity; deoxyhypusine synthase; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Escherichia coli - genetics; Eukaryotic Translation Initiation Factor 5A; Guanine - analogs & derivatives; Guanine - pharmacology; Humans; Hydrogen-Ion Concentration; hypusine; initiation factor eIF-5A; Molecular Weight; NAD - pharmacology; Oxidoreductases Acting on CH-NH Group Donors - antagonists & inhibitors; Oxidoreductases Acting on CH-NH Group Donors - metabolism; Peptide Initiation Factors - metabolism; Protein Binding - drug effects; Protein Precursors - metabolism; Protons; Recombinant Fusion Proteins - metabolism; RNA-Binding Proteins; Sequence Analysis; spermidine; Spermidine - pharmacology; Temperature; Thermodynamics; Ultracentrifugation
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/0264-6021:3400273

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