Inhibition of deoxyhypusine hydroxylase by polyamines and by a deoxyhypusine peptide

The inhibition of deoxyhypusine hydroxylase was studied in vitro. Of the polyamines tested, spermine and its homologue thermine exhibited the strongest inhibition against the enzyme from rat testis. Kinetic analysis revealed that the inhibition by spermine was competitive (Ki, 0.25 +/- 0.02 mM) with respect to the deoxyhypusine protein substrate. Spermidine and its homologue caldine were also inhibitors, but less potent ones than spermine. The spermidine analogues with one or both primary amino groups replaced by the cyano group did not inhibit. A number of diamines, including putrescine, were found to display little or no inhibition. The observed effects of naturally occurring polyamines on deoxyhypusine hydroxylase activity is consistent with a suggestion of regulation of this enzymic activity by cellular levels of polyamines. A synthetic peptide Lys-Thr-Gly-deoxyhypusine-His-Gly-His-Ala-Lys, the amino acid sequence of which corresponds to that surrounding hypusine in eukaryotic initiation factor 4D, was found to display competitive-type inhibition (Ki, 0.44 +/- 0.02 mM) against deoxyhypusine hydroxylase from Chinese hamster ovary cells. Free hypusine and deoxyhypusine, on the other hand, possessed no inhibitory properties. A peptide analogous to the deoxyhypusine nonapeptide with lysine in place of deoxyhypusine had little effect on enzyme activity. The preparation of a derivative of deoxyhypusine, suitably protected for use in the solid-phase synthesis of deoxyhypusine peptides, is described.