Complex formation between deoxyhypusine synthase and its protein substrate, the eukaryotic translation initiation factor 5A (eIF5A) precursor.
Deoxyhypusine synthase catalyses the first step in the post-translational synthesis of hypusine [Nepsilon-(4-amino-2-hydroxybutyl) lysine] in a single cellular protein, the precursor of eukaryotic initiation factor 5A (eIF5A). Deoxyhypusine synthase exists as a tetramer with four potential active sites. The formation of a stable complex between human deoxyhypusine synthase and its protein substrate, human recombinant eIF5A precursor (ec-eIF5A), was examined by affinity chromatography using polyhistidine-tagged (His.Tag) ec-eIF5A, by a gel mobility-shift method, and by analytical ultracentrifugation. Deoxyhypusine synthase was selectively retained by His.Tag-ec-eIF5A immobilized on a resin. The complex of deoxyhypusine synthase and ec-eIF5A was separated from the free enzyme and protein substrate by electrophoresis under non-denaturing conditions. The stoichiometry of the two components in the complex was estimated to be 1 deoxyhypusine synthase tetramer to 1 ec-eIF5A monomer by N-terminal amino acid sequencing of the complex. Equilibrium ultracentrifugation data further supported this 1:1 ratio and indicated a very strong interaction of the enzyme with ec-eIF5A (Kd</=0.5 nM). Formation of the complex was not dependent on NAD+ or spermidine and occurred at pH7.0-9.2. An enzyme-product complex, as well as the deoxyhypusine-containing product (modified ec-eIF5A), was also detected at pH7.0-9.2 in a complete reaction mixture containing 1 mM spermidine.
Lee
Y B
YB
Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892-4340, USA.
Joe
Y A
YA
Wolff
E C
EC
Dimitriadis
E K
EK
Park
M H
MH
D002457:Cell Extracts; D002846:Chromatography, Affinity; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006147:Guanine / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009243:NAD / Q000494:pharmacology; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010448:Peptide Initiation Factors / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D011498:Protein Precursors / Q000378:metabolism*; D011522:Protons; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017421:Sequence Analysis; D013095:Spermidine / Q000494:pharmacology; D013696:Temperature; D013816:Thermodynamics; D014461:Ultracentrifugation; D000097574:Eukaryotic Translation Initiation Factor 5A
D002457:Cell Extracts; D006147:Guanine; D009243:NAD; D000587:Oxidoreductases Acting on CH-NH Group Donors; D010448:Peptide Initiation Factors; D011498:Protein Precursors; D011522:Protons; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D013095:Spermidine; D000097574:Eukaryotic Translation Initiation Factor 5A; C100667:N(1)-guanyl-1,7-diaminoheptane; C063507:deoxyhypusine synthase