Microsomal glutathione transferase : studies on the kinetic mechanism, species variety, binding properties and substrate measurement

Sammanfattning: Microsomal glutathione transferase (MGST) is a membrane bound detoxification enzyme, which has been purified from different species including rat, mouse, human, frog, and fish. The rat MGST1 is localized predominantly in the liver microsomes and the outer mitochondrial membrane. The major function of the enzyme is conjugation of a broad range of hydrophobic xenobiotics with glutathione, which mostly results in detoxification and facilitates excretion. The activity of mammalian MGST1 (towards 1-chloro-2,4-dinitrobenzene, CDNB) could be activated upon the addition of sulfhydryl reagents (N-ethylmaleimide, NEM) and this interesting property has been studied continuously in our laboratory by experiments aimed at determining the kinetic (and functional) mechanism. Using alternate substrate diagnosis, the catalytic mechanism of the microsomal glutathione transferase 1 is defined as a random sequential mechanism. The activation of the enzyme, increasing the catalytic efficiency, was observed not only towards more reactive substances, but also towards less reactive substances such as 4-chloro-3nitrobenzamide when GSH is present at low concentration. The fact that the pKa of the thiol in the enzyme bound GSH (6.4) is lower than the pKa of free GSH (9.2) is an important aspect of the chemical catalysis. No significant change of the pKa value was found between unactivated and activated enzyme. The binding of GSH and an inhibitor to rat liver microsomal glutathione transferase was studied by use of equilibrium dialysis and equilibrium partition in a two-phase system. The radioactive substrate glutathione and an inhibitor (glutathione sulfonate) give hyperbolic binding isotherms with a stoichiometry of 1 mol per 1 mol of enzyme (i.e., 1 molecule bound per homotrimer). Glutathione had an equilibrium binding constant of 17 [my]M. Glutathione sulfonate was used in competition experiments and the results showed that glutathione sulfonate could effectively displace GSH. The Kd and Ki for glutathione sulfonate are both 10 [my]M. We found no change in these parameters between the unactivated and activated enzyme forms (by N-ethylmaleimide). Thus, we can conclude that activation does not result from changes in binding affinity to GSH. New microsomal glutathione transferases were purified from frog and pike liver. The two enzymes displayed extremely high activity towards 1-chloro-2,4-dinitrobenzene (200 times higher than vertebrate microsomal glutathione transferase 1 in unactivated form). Frog and pike liver MGST could be inactivated by NEM as a distinguishing property of the enzyme from lower vertebrates. The molecular mass of the two enzymes is about 17 kDa and the N-terminal amino acid sequence of pike microsomal glutathione transferase is similar to mammalian MGST1 The enzymes displayed glutathione peroxidase activity towards products of lipid peroxidation. Therefore, the suggested function of protection from lipid peroxidation was also identified in lower vertebrates. Traditional inhibitors of the MGST1 could also inhibit these enzymes. In contrast, the frog and pike enzymes displayed some differences in optimum pH range and inactivity towards N-acetyl-L-cysteine. The microsomal GST expressed in pike liver microsomes is estimated to be lower than 0.0022% of the total microsomal protein. This expression level is about 1400 times lower than that of rat liver microsomal GST1. So in lower vertebrates, microsomal GSTs displaying a lower expression level and a higher activity may be a balanced "strategy" in evolution. A simple rapid and sensitive fluorimetric method for the specific determination of reduced glutathione was developed. This method was based on glutathione S-transferase conjugation of GSH to monochlorobimane. Some important features such as the effects of dithioerythritol and the optimal conditions for forming the product GS-bimane were studied. The method is applicable to the determination of GSH in pure samples and in peripheral blood. A detection limit of 1 pmol was achieved. This assay is one of the most sensitive methods so far described. The method can thus be quite useful regarding research on the relationship between GSH levels and toxicity.