Abstract
Deoxyhypusine synthase catalyzes the first step in hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine) synthesis in a single cellular protein, eIF5A precursor. The synthesis of deoxyhypusine catalyzed by this enzyme involves transfer of the 4-aminobutyl moiety of spermidine to a specific lysine residue in the eIF5A precursor protein to form a deoxyhypusine-containing eIF5A intermediate, eIF5A(Dhp). We recently discovered the efficient reversal of deoxyhypusine synthesis. When eIF5A([3H]Dhp), radiolabeled in the 4-aminobutyl portion of its deoxyhypusine residue, was incubated with human deoxyhypusine synthase, NAD, and 1,3-diaminopropane, [3H]spermidine was formed by a rapid transfer of the radiolabeled 4-aminobutyl side chain of the [3H]deoxyhypusine residue to 1,3-diaminopropane. No reversal was observed with [3H]hypusine protein, suggesting that hydroxylation at the 4-aminobutyl side chain of the deoxyhypusine residue prevents deoxyhypusine synthase-mediated reversal of the modification. Purified human deoxyhypusine synthase also exhibited homospermidine synthesis activity when incubated with spermidine, NAD, and putrescine. Thus it was found that [14C]putrescine can replace eIF5A precursor protein as an acceptor of the 4-aminobutyl moiety of spermidine to form radiolabeled homospermidine. The Km value for putrescine (1.12 mM) as a 4-aminobutyl acceptor, however, is much higher than that for eIF5A precursor (1.5 microM). Using [14C]putrescine as an acceptor, various spermidine analogs were evaluated as donor substrates for human deoxyhypusine synthase. Comparison of spermidine analogs as inhibitors of deoxyhypusine synthesis, as donor substrates for synthesis of deoxyhypusine (or its analog), and for synthesis of homospermidine (or its analog) provides new insights into the intricate specificity of this enzyme and versatility of the deoxyhypusine synthase reaction.
Highlights
From the Oral and Pharyngeal Cancer Branch, NIDCR, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892
We have established that deoxyhypusine synthesis occurs by way of a covalent enzyme-imine intermediate [12] in four steps (Scheme 1, left-hand pathway): (i) NAD-dependent dehydrogenation of spermidine to form dehydrospermidine, (ii) transfer of the 4-aminobutyl moiety from dehydrospermidine to the ⑀-amino group of an active site lysine residue (Lys-329 in the human enzyme) to form a covalent enzyme-imine intermediate, (iii) transfer of the 4-aminobutyl moiety from the enzyme-imine intermediate to the N⑀-amino group of a specific lysine residue of the eIF5A precursor (Lys-50 in the human protein [13]) to form an eIF5A-imine intermediate, and (iv) reduction of this intermediate by enzyme-bound NADH to form a deoxyhypusine residue [14]
Reversal of Deoxyhypusine Synthesis Reaction and Identification of Products—When purified eIF5A([3H]Dhp) was incubated with deoxyhypusine synthase in the presence of NAD and 1,3-diaminopropane, a rapid loss of labeled protein was observed with a concomitant increase in trichloroacetic acidsoluble radioactivity (Fig. 1A, lane 5, top panel, radioactivity in protein after SDS-PAGE; lower panel, trichloroacetic acid-soluble radioactivity)
Summary
Previous studies with a large number of compounds structurally related to spermidine as inhibitors of deoxyhypusine synthesis [15,16,17], and the topology of the proposed spermidine-binding pocket of the enzyme [11] revealed a narrow specificity toward the donor substrates, with spermidine being strongly favored. It was not known whether deoxyhypusine synthase can catalyze other reactions in polyamine metabolism or perform ium in the deoxyhypusine moiety; ec-eIF5A, recombinant human eIF5A(Lys) (precursor protein) expressed in E. coli; Dhp, deoxyhypusine; DHS, deoxyhypusine synthase; hDHS, human deoxyhypusine synthase; BSA, bovine serum albumin; DTT, dithiothreitol
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