Cleavage of spermidine as the first step in deoxyhypusine synthesis. The role of NAD.
The biosynthesis of deoxyhypusine (N-(4-aminobutyl)lysine) occurs by the transfer of the 4-aminobutyl moiety of spermidine to a specific lysine residue in a precursor of eukaryotic translation initiation factor 4D (eIF-4D). Deoxyhypusine synthase, the enzyme that catalyzes this reaction, was purified approximately 700-fold from rat testis. The Km values for the substrates, spermidine, the eIF-4-D precursor protein, and NAD+, were estimated as approximately 1, 0.08, and 30 microM, respectively. After incubation of partially purified enzyme with [1,8-3H]spermidine, NAD+, and the eIF-4D precursor, equal amounts of radioactivity were found in free 1,3-diaminopropane and in protein-bound deoxyhypusine. However, when the protein substrate (eIF-4D precursor) was omitted, radioactivity was found in 1,3-diaminopropane and in delta 1-pyrroline in nearly equal quantities, providing evidence that the cleavage of spermidine occurs, albeit at a slower rate, in the absence of the eIF-4D precursor. That NAD+, which is required for this reaction, functions as the hydrogen acceptor was demonstrated by the fact that radioactivity from spermidine labeled with 3H at position 5 is found in NADH as well as in delta 1-pyrroline. Transfer of this hydrogen from spermidine to the re face of the nicotinamide ring of NAD+, as determined by the use of dehydrogenases of known stereospecificity, defines the first step of deoxyhypusine synthesis as a pro-R, or A, stereospecific dehydrogenation. Based on these findings, an enzyme mechanism involving imine intermediate formation is proposed.
Wolff
E C
EC
Laboratory of Cellular Development and Oncology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892.
Park
M H
MH
Folk
J E
JE
D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D007700:Kinetics; D008239:Lysine / Q000031:analogs & derivatives* / Q000096:biosynthesis; D008297:Male; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000302:isolation & purification / Q000378:metabolism*; D010448:Peptide Initiation Factors / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism; D016601:RNA-Binding Proteins; D051381:Rats; D013095:Spermidine / Q000378:metabolism*; D013737:Testis / Q000201:enzymology*; D000097574:Eukaryotic Translation Initiation Factor 5A
D008239:Lysine; D009243:NAD; D000587:Oxidoreductases Acting on CH-NH Group Donors; D010448:Peptide Initiation Factors; D011498:Protein Precursors; D016601:RNA-Binding Proteins; D013095:Spermidine; D000097574:Eukaryotic Translation Initiation Factor 5A; C034924:deoxyhypusine; C063507:deoxyhypusine synthase