Identification of lysine(350) of yeast deoxyhypusine synthase as the site of enzyme intermediate formation

The posttranslational formation of deoxyhypusine in the precursor of eukary otic initiation factor 5A (eIF5A) is catalysed by deoxyhypusine synthase. T his NAD-dependent reaction involves transfer of the 4-aminobutyl moiety of spermidine to a single lysine residue in the eIF5A precursor. The present s tudy shows evidence for the formation of a covalent enzyme-substrate interm ediate 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 enzym e intermediate and consequently precluded deoxyhypusine synthesis in the eI F5A precursor, leading to the conclusion that the enzyme intermediate forme d at Lys(350) is critical for deoxyhypusine synthesis activity. The results provide a rational basis for the inability of the mutated deoxyhypusine sy nthase gene encoding arginine in place of Lys(350) to support growth in yea st (Park; er al., 1998). The demonstration of the formation of an enzyme-im ine intermediate in yeast deoxyhypusine synthase analogous to that of the h uman enzyme strongly suggests that the enzyme mechanism is conserved in div erse eukaryotes. Copyright (C) 1999 John Wiley & Sons, Ltd.