Evaluation of the metal ion requirement of the human deoxyhypusine hydroxylase from HeLa cells using a novel enzyme assay.
Hypusine synthesis in the eukaryotic initiation factor 5A is a unique two-step posttranslational modification. After deoxyhypusine is generated by the deoxyhypusine synthase, the deoxyhypusine hydroxylase (EC 1.14.99.29) catalyzes the formation of mature hypusine. A rapid assay for monitoring the deoxyhypusine hydroxylase activity was established, employing the oxidative cleavage of the hypusyl residue and subsequent extraction of the generated aldehydes. As metal ion chelators have been reported to inhibit the deoxyhypusine hydroxylase, the mechanism of this inhibition and the effect of transition metal ions on enzyme activity were investigated. A ferric ion appears to be essential for enzymatic activity, the inhibition of which is entirely attributed to the metal ion binding capacity of the chelators.
Csonga
R
R
Sandoz Research Institute, Department of Immunodermatology, Vienna, Austria.
Ettmayer
P
P
Auer
M
M
Eckerskorn
C
C
Eder
J
J
Klier
H
H
D002614:Chelating Agents / Q000494:pharmacology; D003035:Cobalt / Q000494:pharmacology; D003300:Copper / Q000494:pharmacology; D005290:Ferric Compounds / Q000494:pharmacology; D005296:Ferrous Compounds / Q000494:pharmacology; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D008239:Lysine / Q000031:analogs & derivatives / Q000096:biosynthesis; D008345:Manganese / Q000494:pharmacology; D008670:Metals / Q000494:pharmacology*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009532:Nickel / Q000494:pharmacology; D010084:Oxidation-Reduction; D010448:Peptide Initiation Factors / Q000378:metabolism; D016601:RNA-Binding Proteins; D015032:Zinc / Q000494:pharmacology; D000097574:Eukaryotic Translation Initiation Factor 5A
D002614:Chelating Agents; D003035:Cobalt; D003300:Copper; D005290:Ferric Compounds; D005296:Ferrous Compounds; D008239:Lysine; D008345:Manganese; D008670:Metals; D006899:Mixed Function Oxygenases; D009532:Nickel; D010448:Peptide Initiation Factors; D016601:RNA-Binding Proteins; D015032:Zinc; D000097574:Eukaryotic Translation Initiation Factor 5A; C100028:hypusine; C047991:deoxyhypusine hydroxylase