| Range |
0.0081 Sec^-1
|
| Organism |
Human Homo sapiens |
| Reference |
Lee MD, Antczak C, Li Y, Sirotnak FM, Bornmann WG, Scheinberg DA. A new human peptide deformylase inhibitable by actinonin. Biochem Biophys Res Commun. 2003 Dec 12 312(2):309-15 Table - link PubMed ID14637138
|
| Method |
Cloning of HsPDF cDNA. Expression and purification of HsPDF. The expression vectors encoding the HsPDF cDNAs were transformed into BL21(DE3)pLysS cells (Novagen) and grown in LB media containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol at 37 °C until OD600 reached 0.4. Cells were then induced with 0.5 mM IPTG and incubated at 30 °C for 18 h. Peptide deformylase assays. Three different assays were used to study the kinetic properties of HsPDF. Method I is an indirect spectrophotometric assay [15] that couples peptide deformylase activity with formate dehydrogenase (FDH) activity. Method II is a continuous spectrophotometric assay for peptide deformylase activity[16] that uses fML-pNA as the substrate, which is first deformylated and then subsequently processed by Aeromonas proteolytica aminopeptidase (AAP) to release p-nitroaniline. Method III is a direct solid phase fluorescent assay to measure peptide deformylase activity and is based on a previously published method using fluorescamine to detect the formation of the new amino group of the N-formylated substrate after deformylation. In order to examine some standard enzymatic parameters, researchers used the cobalt-containing form of HsPDF to conduct basic kinetic studies. They examined the use of different formylated peptides as potential substrates and found that HsPDF was able to deformylate fMAS, fML-pNA, but not fMLP (Fig. 2 and Table 1). They found that at concentrations above 20 mM of fMAS, the substrate inhibited the human enzyme, an observation that has been noted previously with EcPDF [20]. The Kcat/Km values for HsPDF were significantly lower than the published EcPDF, AtPDF1A, and PfPDF values |
| Comments |
Peptide deformylase (PDF) activity was thought to be limited to ribosomal protein synthesis in prokaryotes, where new peptides are initiated with an N-formylated methionine. It has since been discovered in eukaryotes including Thale cress (arabidopsis) and humans. For Kcat of PDF in E. coli, P. falciparium and A. thaliana see table link, BNID 104974 and a different value for E. coli at BNID 104972 |
| Entered by |
Uri M |
| ID |
104975 |