Haem peptide–protein interactions. Part 4.—The microperoxidase-8-mediated inhibition of the human placental glutathione S-transferase π catalysed conjugation of glutathione and 1-chloro-2,4-dinitrobenzene

Abstract

The inhibition of the glutathione S-transferase π(GST) catalysed conjugation of glutathione (GSH) with 1-chloro-2,4-dinitrobenzene (CDNB) by the haem octapeptide microperoxidase-8 (MP-8) has been investigated. Incubation of the enzyme with MP-8 results in a pseudo-first-order partial inactivation of the enzyme [k_obs= 2.3 (±0.4)× 10^–3 s^–1; pH 6.5; 22.5 °C]. The k_obs is identical to that found for dilutional inactivation of the enzyme observed in buffer alone. MP-8 increases only the extent of inactivation above that observed for dilutional inactivation.

Incubation of the enzyme with varying concentrations of MP-8 was followed by a steady-state kinetic study of the enzyme-catalysed conjugation of GSH with CDNB at (a) fixed [GSH], varying [CDNB] and (b) fixed [CDNB] varying [GSH]. In case (a) the data conformed closely with mixed competitive/non-competitive inhibition kinetics (K_i= 3.2 × 10^–7 mol dm^–3, pH 6.5, 30 °C), while in case (b) the extent of inhibition was insufficient to allow analysis.

These observations are rationalized and discussed in the light of the kinetic studies of MP-8 binding to GST π reported in the preceding paper.