Figure 1.
Synthesis of peroxytetradecanoic acid.
The peroxytetradecanoic acid is formed in the reaction of tetradecanoic acid with hydrogen peroxide.
Figure 2.
The enzymatic activity of PTP CD45.
The effect of tetradecanoic acid, peroxytetradecanoic acid and H2O2 on enzymatic activity of PTP CD45 before and after treatment with DTT. The enzyme CD45 (130 nM) was incubated with 50 nM tetradecanoic acid, peroxytetradecanoic acid and H2O2 at 37°C for 15 minutes. The activity was measured after adding 1 mM pNPP in 50 mM Tris buffer, pH 7.4. After the following 5 min of incubation, PTP activity was measured using microplate reader at 405 nm. Subsequently 20 mM dithiothreitol (DTT) was added to each sample for 30 minutes and a potential recovery of the enzymatic activity was assessed using the same technique. The results were expressed as percent of activity of control sample in Tris buffer. The data from three independent experiments were present as mean ± S.E.M; * significantly different (P<0.05) from control, ** significantly different (P<0.001) from control. The data were analysed using the combination of ANOVA and Tukey’s test (GraphPad Prism Software v.4).
Figure 3.
Oxidation steps of PTP catalytic cysteine residue.
The cysteine residue exists in a thiolate anion form and may undergo oxidation to the inactive sulfenic acid residue form. This conversion is reversible, but highly oxidizing conditions can further induce oxidation to the sulfinic and sulfonic acid residues, which is considered irreversible under physiological conditions.
Table 1.
The calculated binding affinities to CD45 catalytic center.
Figure 4.
The best predicted binding poses of ligands in the CD45 active site.
The best predicted binding poses for peroxy acid C14 (panel A) and hydrogen peroxide (panel B) in the CD45 active site. The receptor was based on the CD45 D1 domain from PDB structure 1YGU [20], with residues mutated to correspond to a CD45 reference sequence (UniProtKB accession number P08575) using the SWISS-MODEL web server [11]. The docking was performed with the AutoDock Vina version 1.1.1 software [15] using a rigid receptor and a binding box centered on the CD45 phosphatase active site. The best binding pose was defined as the pose with the strongest affinity in the largest cluster of poses, with poses clustered with a 1.5 Å RMSD thresholds. Also highlighted are four important residues involved in binding (Tyr683, His822, Arg859 and Gln897) and the catalytic cysteine (Cys853). Predicted hydrogen bonds with a 3.5 Å distance cutoff are shown as green dashed lines. The residues are numbered according the P08575 sequence.