Catalytic studies of glutathione transferase from Clarias gariepinus (Burchell) in dilute and crowded solutions

Abstract

Kinetic properties of purified Clarias gariepinus glutathione transferase (CgGST) was studied in the presence of Ficoll 70, Polyethylene glycol (PEG) 6000, bovine serum albumin (BSA) and in dilute solution. This was done to mimic the cytosol thereby unraveling the actual mechanism of detoxication involving glutathione transferase (GST) in the crowded intracellular milieu. CgGST from the liver of Clarias gariepinus was purified to homogeneity by affinity chromatography on glutathione (GSH) - agarose. Initial-velocity study was performed by varying the concentrations of GSH at various fixed concentrations of 1-chloro-2,4-dinitrobenzene (CDNB) and vice-versa. Data obtained were fitted to the three equations representing random-ordered, compulsory-ordered and ping-pong mechanisms to obtain kinetic parameters. Product inhibition studies using sodium chloride (NaCl) was done by varying the concentrations of NaCl and CDNB at a fixed concentration of GSH and vice-versa. Data obtained were fitted to three equations representing competitive, non-competitive and uncompetitive inhibitions to obtain the inhibition constants (KiGSH and KiCDNB). Optimal temperature of CgGST activity was 20 °C both in dilute and crowded solutions. Maximum velocity (Vmax) in dilute solution was decreased, while KmGSH and KmCDNB were increased in the presence of the crowding agents. Turnover number (kcat), catalytic efficiency - kcat/KmGSH,kcat/KmCDNB and inhibition constants – (KiGSH and KiCDNB) were reduced in crowded solutions. Mechanism of catalysis was steady – state random sequential in both dilute and crowded solutions. The study concluded that although the catalytic efficiency of the enzyme was reduced in crowded solution, mechanism of catalysis remains the same in both crowded and dilute solutions.