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

Biochem J. 1999 May 15;340 ( Pt 1)(Pt 1):273-81.

Abstract

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.

MeSH terms

  • Cell Extracts
  • Chromatography, Affinity
  • Dose-Response Relationship, Drug
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / genetics
  • Guanine / analogs & derivatives
  • Guanine / pharmacology
  • Humans
  • Hydrogen-Ion Concentration
  • 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
  • RNA-Binding Proteins*
  • Recombinant Fusion Proteins / metabolism
  • Sequence Analysis
  • Spermidine / pharmacology
  • Temperature
  • Thermodynamics
  • Ultracentrifugation

Substances

  • Cell Extracts
  • N(1)-guanyl-1,7-diaminoheptane
  • Peptide Initiation Factors
  • Protein Precursors
  • Protons
  • RNA-Binding Proteins
  • Recombinant Fusion Proteins
  • eukaryotic translation initiation factor 5A
  • NAD
  • Guanine
  • Oxidoreductases Acting on CH-NH Group Donors
  • deoxyhypusine synthase
  • Spermidine