STEADY-STATE KINETICS AND CHEMICAL MECHANISM OF OCTOPUS HEPATOPANCREATIC GLUTATHIONE TRANSFERASE

The kinetic mechanism of glutathione S-transferase (GST) from Octopus vulgaris hepatopancreas was investigated by steady-state analysis. Initial-velocity studies showed an intersecting pattern, which suggests a sequential kinetic mechanism for the enzyme. Product-inhibition patterns by chloride and the conjugate product were all non-competitive with respect to glutathione or 1-chloro-2,4-dinitrobenzene (CDNB), which indicates that the octopus digestive gland GST conforms to-a steady-state sequential random Bi Bi kinetic mechanism. Dead-end inhibition patterns indicate that ethacrynic acid {[2,3;dichloro 4-(2-methylenebutyryl) phenoxy]acetic acid} binds at the hydrophobic H-site, norophthalmic acid (gamma-glutamylalanylglycine) binds at the glutathione G-site, and glutathione-ethacrynate conjugate occupied both H- and G-sites of the enzyme. The chemical mechanism of the enzyme was examined by pH and kinetic solvent-isotope effects. At pH (and p(2)H) = 8.011, in which k(cat.) was independent of pH or p(2)H, the solvent isotope effects on V and V/K-mGSH were near unity, in the range 1.069-1.175. An inverse isotope effect was observed for V/K-mCDNB (0.597), presumably resulting from the hydrogen-bonding of enzyme-bound glutathione, which has pK(a) of 6.83 +/- 0.04, a value lower by 2.34 pH units than the pK(a) of glutathione in aqueous solution. This lowering of the pK(a) value for the sulphydryl group of the bound glutathione was presumably due to interaction with the active site Tyr(7), which had a pK(a) value of 8.46 +/- 0,09 that was raised to 9.63 +/- 0.08 in the presence of glutathione thiolate. Subsequent chemical reaction involves attacking of thiolate anion at the electrophilic substrate with the formation of a negatively charged Meisenheimer complex, which is the rate-limiting step of the reaction.