Ellagic acid-mediated inhibition of schistosoma japanicum glutathione transferase: enzyme kinetics and structural perspectives

Abstract

Schistosoma japonicum glutathione S-transferase (SjGST) is a homodimeric enzyme central to detoxification of electrophilic compounds in the parasite due to its lack of cytochrome P-450. Hence, SjGST is a potential therapeutic target for schistosomiasis. Akin to most cytosolic GSTs, SjGST is composed of two active sites per monomer, which are the glutathione binding site (G-site) and the hydrophobic binding site (H-site). Ellagic acid (EA), a polyphenolic compound ubiquitous in fruits and other plant-based products has been identified as a therapeutic agent against cancer, heart and liver diseases. The interaction between SjGST and EA was characterised in this study. Using 1-chloro-2,4 dinitrobenzene (CDNB) and glutathione (GSH) as SjGST substrates, EA was shown to inhibit SjGST activity, with an IC50 of 2.4 μM. The observed mode of inhibition with respect to the G-site and H-site was non-competitive and uncompetitive, respectively. The turnover rate of SjGST was reduced from 0.002 ± 0.0001 sec1 to 0.0006 ± 0.00001 sec-1 in the presence of 9 μM EA, while the catalytic efficiency (kcat/KM) was reduced from 0.02 ± 0.0002 M-1s-1 to 0.009 ± 0.0001 M-1s-1. Using ANS as an extrinsic fluorescence probe, SjGST bound to ANS in a concentration-dependent manner in the absence (Kd = 40.6 ± 2.6 μM) or presence of EA (Kd = 30.3 ± 1.4 μM). This observation was further probed by the displacement of ANS by EA, which suggest that about 64.5% of ANS was displaced by EA. The ANS studies indicated that EA binds to the H-site of the enzyme. Secondary and quaternary structure analyses indicate that EA does not alter the secondary and tertiary structures of SjGST. Thermodynamic parameters obtained by isothermal titration calorimetry (ITC) indicate that the interaction between SjGST and EA is enthalpically-driven and compensated by a negative entropy change (-T∆S = 20.40 ± 0.08 kJ/mol) because of enthalpy-entropy compensation. The free energy change (∆G = -29.88 ± 0.07 kJ/mol) is also favoured by entropic contributions; therefore, indicating a strong hydrophobic interaction. A stoichiometry of 4 molecule of EA per mole of the dimeric enzyme was obtained. Molecular modelling studies suggests that EA binds more to the dimer interface compared with the Hsite. Therefore, it is possible that EA may bind to two different sites in the enzyme, the H-site and the dimer interface.