E. Wolff, J. E. Folk, M. Park
Jun 20, 1997
Citations
1
Influential Citations
43
Citations
Journal
The Journal of Biological Chemistry
Abstract
Deoxyhypusine (N ε-(4-aminobutyl)lysine) is the key intermediate in the posttranslational synthesis of the unique amino acid, hypusine (N ε-(4-amino-2-hydroxybutyl)lysine). Deoxyhypusine synthase catalyzes the formation of deoxyhypusine by conjugation of the butylamine moiety of spermidine to the ε-amino group of one specific lysine residue of the eukaryotic translation initiation factor 5A (eIF-5A) precursor protein. However, in the absence of the eIF-5A precursor, catalysis involves only the NAD-dependent cleavage of spermidine to generate 1,3-diaminopropane and a putative 4-carbon amine intermediate that gives rise to Δ1-pyrroline. We have obtained evidence for a covalent enzyme-substrate intermediate that accumulates in the absence of the eIF-5A precursor. Incubation of human recombinant enzyme with [1,8-3H]spermidine and NAD, followed by reduction with NaBH3CN, resulted in specific radiolabeling of the enzyme. The radioactive component in the reduced enzyme intermediate was identified as deoxyhypusine and was shown to occur at a single locus. The fact that labeled deoxyhypusine was found after treatment with a reducing agent suggests an intermediate with the butylamine moiety derived from spermidine attached through an imine linkage to the ε-amino group of a specific lysine residue of the enzyme. This residue has been identified as lysine 329. Separate experiments showing efficient transfer of labeled butylamine moiety from enzyme intermediate to eIF-5A precursor strongly support a reaction mechanism involving an imine intermediate.