Cleavage of Spermidine as the First Step in Deoxyhypusine Synthesis. The Role of NAD

J Biol Chem. 1990 Mar 25;265(9):4793-9.

Abstract

The biosynthesis of deoxyhypusine (N-(4-aminobutyl)lysine) occurs by the transfer of the 4-aminobutyl moiety of spermidine to a specific lysine residue in a precursor of eukaryotic translation initiation factor 4D (eIF-4D). Deoxyhypusine synthase, the enzyme that catalyzes this reaction, was purified approximately 700-fold from rat testis. The Km values for the substrates, spermidine, the eIF-4-D precursor protein, and NAD+, were estimated as approximately 1, 0.08, and 30 microM, respectively. After incubation of partially purified enzyme with [1,8-3H]spermidine, NAD+, and the eIF-4D precursor, equal amounts of radioactivity were found in free 1,3-diaminopropane and in protein-bound deoxyhypusine. However, when the protein substrate (eIF-4D precursor) was omitted, radioactivity was found in 1,3-diaminopropane and in delta 1-pyrroline in nearly equal quantities, providing evidence that the cleavage of spermidine occurs, albeit at a slower rate, in the absence of the eIF-4D precursor. That NAD+, which is required for this reaction, functions as the hydrogen acceptor was demonstrated by the fact that radioactivity from spermidine labeled with 3H at position 5 is found in NADH as well as in delta 1-pyrroline. Transfer of this hydrogen from spermidine to the re face of the nicotinamide ring of NAD+, as determined by the use of dehydrogenases of known stereospecificity, defines the first step of deoxyhypusine synthesis as a pro-R, or A, stereospecific dehydrogenation. Based on these findings, an enzyme mechanism involving imine intermediate formation is proposed.

MeSH terms

  • Animals
  • Chromatography, Gel
  • Chromatography, Ion Exchange
  • Kinetics
  • Lysine / analogs & derivatives*
  • Lysine / biosynthesis
  • Male
  • NAD / metabolism*
  • Oxidation-Reduction
  • Oxidoreductases Acting on CH-NH Group Donors / isolation & purification
  • Oxidoreductases Acting on CH-NH Group Donors / metabolism*
  • Peptide Initiation Factors / metabolism
  • Protein Precursors / metabolism
  • RNA-Binding Proteins*
  • Rats
  • Spermidine / metabolism*
  • Testis / enzymology*

Substances

  • Peptide Initiation Factors
  • Protein Precursors
  • RNA-Binding Proteins
  • eukaryotic translation initiation factor 5A
  • NAD
  • deoxyhypusine
  • Oxidoreductases Acting on CH-NH Group Donors
  • deoxyhypusine synthase
  • Lysine
  • Spermidine