In the inhibitory kinetic studies five different concentrati
In the inhibitory kinetic studies, five different concentrations of DEL carefully selected from the near-linear region of the dose–response curve (0.5 μM, 1.0 μM, 2.0 μM, 4.0 μM, and 8.0 μM) were used. The Vm values at fixed [CDNB]–varied [GSH] and at fixed [GSH]–varied [CDNB] were 10.4 ± 0.22 U mg−1 protein and 8.70 ± 0.33 U mg−1 protein, respectively (Figs. 2A and 3A). The results show no significant difference between the Vm values at fixed [CDNB]–varied [GSH] and at fixed [GSH]–varied [CDNB], suggesting that, at the concentrations studied, the rate of reaction and turnover are similar in both cases. On the other hand, the Km values at fixed [CDNB]–varied [GSH] and at fixed [GSH]–varied [CDNB] were 0.31 ± 0.02 mM and 0.30 ± 0.03 mM, respectively (Figs. 2B and 3B). These findings indicate that the affinity of the enzyme for both substrates is almost the same. The entire set of kinetic parameters are summarized in Table 1. The G-site of hpGSTP1-1 is conserved and very specific for GSH only, with Tyr7 residue shown to be the catalytic residue (Board and Menon, 2013; Prade et al., 1997; Oakley et al., 1999). Different from the G-site, the H-site is not well conserved and has broad specificity to permit the binding of a wide range of xenobiotics or electrophiles (Prade et al., 1997). Our kinetic measurements showed that the type of inhibition at both fixed [CDNB]–varied [GSH] and fixed [GSH]–varied [CDNB] was noncompetitive (Figs. 2A and 3A), with Ki values from the statistical analysis being in good agreement with those obtained from the graphical solution (Figs. 2B and 3B). The observed type of inhibition suggests that DEL may have bound to a site other than the CDNB- or GSH-binding site to inhibit hpGSTP1-1 allosterically. Our findings corroborate the long-established characteristic binding of non-substrate ligands to GST Fmoc-D-Phe(4-Cl)-OH with a noncompetitive mode of inhibition in the presence of CDNB and GSH as substrates (Ketley et al., 1975). The binding of DEL to hpGSTP1-1 may have caused conformational changes particularly to the substrate-binding sites of the enzyme such that the enzyme was not able to transform the two substrates (GSH and CDNB) effectively, thus resulting in the observed inhibitory effect. The highest-ranked (calculated binding energy = -7.90 kcal mol−1) docking solution revealed that DEL binds on hpGSTP1-1 at the interface of the two monomers (Fig. 4A), further supporting the notion that it inhibits the enzyme noncompetitively (as observed in our in vitro studies). In this intermonomeric cavity, DEL seemed to be anchored well through establishing two different types of noncovalent interactions, namely hydrophobic and hydrogen-bonding interactions, with the protein subunits labeled A and B (Fig. 4B). The DEL molecule, using its second ring structure, makes two hydrophobic contacts with Tyr49A and another with Asp98B (distances < 4.00 Å). The hydrogen bond is between the amide group of Lys127B and the nitrile group of the DEL molecule (distance = 3.38 Å; donor angle = 107.55°). This finding is in good agreement with the reported molecular electrostatic potential values (in kJ mol−1) located on the cyano, ether, ester and bromine moieties of DEL, which provided a qualitative ranking of the possible intermolecular interactions of different DEL fragments (Taillebois et al., 2015). The same study demonstrated that the main interacting functional groups in order of reactivity were the nitrile group, followed by the carbonyl group, and oxygen atoms of the ether groups. The bromine atoms of the DEL molecule also hold the potential of donating halogen bonds (Taillebois et al., 2015). It is worth mentioning that our computational analysis additionally indicated DEL’s ability to bind to a second cavity on the enzyme (also with a predicted binding energy of -7.90 kcal mol−1) that symmetrically corresponds to the first cavity (data not shown).