Complex formation between deoxyhypusine synthase and its protein substrate, the eukaryotic translation initiation factor 5A (elF5A) precursor

Deoxyhypusine synthase catalyses the first step in the posttranslational sy nthesis of hypusine [N-6-(4-amino-2-hydroxybutyl) lysine] in a single cellu lar protein, the precursor of eukaryotic initiation factor 5A (eIF5A). Deox yhypusine synthase exists as a tetramer with four potential active sites. T he formation of a stable complex between human deoxyhypusine synthase and i ts protein substrate, human recombinant eIF5A precursor (ec-eIF5A), was exa mined by affinity chromatography using polyhistidine-tagged (His Tag) ec-eI F5A, by a gel mobility-shift method, and by analytical ultracentrifugation, Deoxyhypusine synthase was selectively retained by His Tag-ec-eIF5A immobi lized on a resin. The complex of deoxyhypusine synthase and ec-eIF5A was se parated 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 ul tracentrifugation data further supported this 1:1 ratio and indicated a ver y strong interaction of the enzyme with ec-eIF5A (K-d less than or equal to 0.5 nM). Formation of the complex was not dependent on NAD(+) or spermidin e and occurred at pH 7.0-9.2. An enzyme-product complex, as well as the deo xyhypusine-containing product (modified ec-eIF5A), was also detected at pH 7.0-9.2 in a complete reaction mixture containing 1 mM spermidine.