The posttranslational formation of deoxyhypusine in the precursor of eukaryotic initiation factor 5A (eIF5A) is catalysed by deoxyhypusine synthase. This NAD-dependent reaction involves transfer of the 4-aminobutyl moiety of spermidine to a single lysine residue in the eIF5A precursor. The present study shows evidence for the formation of a covalent enzyme-substrate intermediate between a specific lysine residue (Lys 350 ) of yeast deoxyhypusine synthase and the 4-aminobutyl moiety from spermidine. Substitution of this lysine residue with Arg or Ala totally prevented the formation of the enzyme intermediate and consequently precluded deoxyhypusine synthesis in the eIF5A precursor, leading to the conclusion that the enzyme intermediate formed at Lys 350 is critical for deoxyhypusine synthesis activity. The results provide a rational basis for the inability of the mutated deoxyhypusine synthase gene encoding arginine in place of Lys 350 to support growth in yeast (Park et al., 1998). The demonstration of the formation of an enzyme-imine intermediate in yeast deoxyhypusine synthase analogous to that of the human enzyme strongly suggests that the enzyme mechanism is conserved in diverse eukaryotes.