Human deoxyhypusine synthase: interrelationship between binding of NAD and substrates.
Deoxyhypusine synthase catalyses the NAD-dependent transfer of the butylamine moiety from the polyamine, spermidine, to a specific lysine residue of a single cellular protein, eukaryotic translation-initiation factor 5A (eIF5A) precursor. The native enzyme exists as a tetramer of four identical subunits and contains four binding sites for NAD. The binding of spermidine and NAD was studied by a filtration assay. [(3)H]Spermidine binding to the enzyme was not detectable alone or in the presence of the eIF5A precursor, but was detected only in the presence of NAD or NADH, suggesting that a NAD/NADH-induced conformational change is required for the binding of spermidine. A strong NAD-dependent binding was also observed with a spermidine analogue, N(1)-guanyl-1, 7-diamino[(3)H]heptane (GC(7)), but not with [(14)C]putrescine or [(14)C]spermine. Although [(3)H]NAD binding to the enzyme occurred in the absence of spermidine, its affinity for the enzyme was markedly enhanced by spermidine, GC(7) and also by the eIF5A precursor. The maximum binding for NAD and spermidine was estimated to be approximately 4 molecules each/enzyme tetramer. The dependence of spermidine binding on NAD and the modulation of binding of NAD by spermidine and the eIF5A precursor suggest intricate relationships between the binding of cofactor and the substrates, and provide new insights into the reaction mechanism.
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Building 30, Room 211, Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892-4340, USA.
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D001665:Binding Sites; D040061:Eukaryotic Initiation Factor-5; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000031:analogs & derivatives* / Q000378:metabolism; D009243:NAD / Q000378:metabolism*; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000378:metabolism*; D010448:Peptide Initiation Factors / Q000378:metabolism; D011485:Protein Binding; D011498:Protein Precursors / Q000378:metabolism; D013095:Spermidine / Q000031:analogs & derivatives / Q000378:metabolism*; D013379:Substrate Specificity; D013997:Time Factors
D040061:Eukaryotic Initiation Factor-5; D010448:Peptide Initiation Factors; D011498:Protein Precursors; D009243:NAD; C100028:hypusine; D000587:Oxidoreductases Acting on CH-NH Group Donors; C063507:deoxyhypusine synthase; D008239:Lysine; D013095:Spermidine