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Human deoxyhypusine synthase: interrelationship between binding of NAD and substrates
1Building 30, Room 211, Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892-4340, USA.
The Biochemical Journal
|December 6, 2000
Summary
Deoxyhypusine synthase requires NAD or NADH for spermidine binding, indicating a conformational change is necessary. Spermidine and the eIF5A precursor enhance NAD binding, revealing intricate substrate-cofactor interactions in this enzyme.
Area of Science:
- Biochemistry
- Enzymology
- Protein Modification
Background:
- Deoxyhypusine synthase (DHPS) catalyzes a crucial post-translational modification.
- This modification involves the transfer of a butylamine group from spermidine to the eukaryotic translation-initiation factor 5A (eIF5A) precursor.
- The enzyme is a tetramer with four NAD binding sites, and its activity is NAD-dependent.
Purpose of the Study:
- To investigate the binding mechanisms of spermidine and NAD to deoxyhypusine synthase.
- To elucidate the role of cofactor binding in substrate recognition and conformational changes.
- To understand the interplay between NAD, spermidine, and the eIF5A precursor during catalysis.
Main Methods:
- Filtration assays were employed to study the binding kinetics of radiolabeled ligands.
- The binding of [(3)H]spermidine, N(1)-guanyl-1, 7-diamino[(3)H]heptane (GC(7)), [(14)C]putrescine, and [(14)C]spermine was assessed in the presence and absence of NAD, NADH, and the eIF5A precursor.
- The binding affinity of [(3)H]NAD was evaluated under various conditions.
Main Results:
- [(3)H]Spermidine binding was undetectable without NAD or NADH, suggesting a conformational change induced by the cofactor.
- NAD/NADH-induced spermidine binding was confirmed, with a spermidine analogue (GC(7)) also showing strong NAD-dependent binding.
- Spermidine, GC(7), and the eIF5A precursor significantly enhanced the binding affinity of [(3)H]NAD to the enzyme.
- Approximately four molecules of NAD and spermidine bound per enzyme tetramer.
Conclusions:
- Spermidine binding to deoxyhypusine synthase is dependent on the presence of NAD or NADH, implying a cofactor-induced conformational change.
- The eIF5A precursor and spermidine modulate NAD binding, highlighting complex interactions between substrates and cofactors.
- These findings offer new insights into the catalytic mechanism of deoxyhypusine synthase and the regulation of eIF5A modification.