The biosynthesis of protein-bound hypusine (N epsilon -(4-amino-2-hydroxybutyl)lysine). Lysine as the amino acid precursor and the intermediate role of deoxyhypusine (N epsilon -(4-aminobutyl)lysine)

Abstract

The major labeled constituent produced in cellular protein during the incubation of Chinese hamster ovary (CHO) cells with [3H]putrescine or [terminal methylenes-3H]spermidine was identified as hypusine (N epsilon -(4-amino-2-hydroxybutyl)lysine). This unusual amino acid was found to occur predominantly in one relatively acidic low molecular weight protein. When CHO cells were labeled with [4,5-3H)lysine, a small portion of the radioactivity of the cellular protein fraction, after release by proteolytic digestion or acid hydrolysis, chromatographed at the position of hypusine. Oxidative degradation of this isolated labeled material yielded labeled lysine, thus, providing evidence that lysine is the amino acid precursor of hypusine. Upon incubation of CHO cells with the metal chelator, alpha,alpha-dipyridyl, and either [4,5]3H]lysine or [terminal methylenes-3H]spermidine, label was incorporated into a protein-bound material, the chromatographic properties of which, after release by digestion, were found to be different from those of hypusine. This constituent of cell protein was identified as the unhydroxylated form of hypusine, deoxyhypusine (N epsilon -(4-aminobutyl)lysine). Evidence that the normal biosynthesis of hypusine proceeds through hydroxylation of deoxyhypusine was obtained by demonstration of conversion of protein-bound deoxyhypusine to protein-bound hypusine both in intact cells and in cell-free lysate. In the presence of the metal chelator, alpha,alpha-dipyridyl, deoxyhypusine accumulated in a single protein whose two dimensional electrophoretic properties were indistinguishable from those of the usual hypusine-containing protein. This finding supports the proposed mechanism in which peptide-bound lysine is converted to peptide-bound hypusine through hydroxylation of the transitory intermediate, deoxyhypusine.