IDENTIFICATION OF N-ACETYLCYSTEINE AS A NEW SUBSTRATE FOR RAT-LIVER MICROSOMAL GLUTATHIONE TRANSFERASE - A STUDY OF THIOL LIGANDS

N-Acetyl-L-cysteine serves as an efficient substrate for the rat liver microsomal glutathione transferase with 1-chloro-2,4-dinitrobenzene a s second substrate (8.8 +/-0.37 mumol/min mg). The activity is actuall y higher than that obtained with glutathione (2-4 mumol/min mg). In ex amining the activity of liver subcellular fractions, no activity with N-acetyl-L-Cys could be detected in dialyzed or N-ethylmaleimide-treat ed (in order to remove endogenous glutathione) cytosol. The activity i n rat liver microsomes was 0.11 +/- 0.007 mumol/min mg, which is accou nted for by the content of microsomal glutathione transferase. Thus, N -acetyl-L-Cys can be used as a specific substrate for determining the conjugating activity of microsomal glutathione transferase. N-Acetyl-L -Cys was also shown to function as a substrate for the enzyme when oth er second substrates than 1-chloro-2,4-dinitrobenzene (with varying el ectrophilicity) are used. The pH dependence of microsomal glutathione transferase was studied. The k(cat)/K(m(1-chloro-2,4-dinitrobenzene)) was dependent on pH with an apparent pK(a) of 6, greater-than-or-equal -to 9, and greater-than-or-equal-to 8 with saturating glutathione, gam ma-L-Glu-L-Cys, and N-acetyl-L-cysteine, respectively. Apparently the enzyme has the ability to lower the pK(a) of glutathione by 3 orders o f magnitude. The k(cat)/K(m(thiol) did not vary appreciably with pH (e xcept for N-acetyl-L-cysteine), indicating that no rate-determining de protonation occurs on the enzyme itself between pH 5.5 and 9. The abil ities of histidine-, lysine-, and arginine-selective reagents to inact ivate the enzyme when N-acetyl-L-cysteine and gamma-L-Glu-L-Cys were u sed as substrates were investigated. The activity toward N-acetyl-L-cy steine was decreased considerably less after treatment with the argini ne-selective reagent phenylglyoxal, as compared to the activity toward GSH andy-L-Glu-L-Cys. This indicates that an arginine makes contact w ith the gamma-L-Glu residue in GSH. With the other reagent/substrate c ombinations tested the enzyme was inactivated almost completely. The a bility of microsomal glutathione transferase to stabilize the Meisenhe imer complex formation between 1,3,5-trinitrobenzene and various gluta thione analogues, including non-substrate thiols, has been examined. I t is shown that, in general, substrates exhibited higher formation con stants (approaching 50 mM-1) than non-substrates (4.5 +/- 1.7 mM-1, n = 7), whereas simpler thiols did not yield enzyme-bound complexes. The fact that the enzyme can stabilize Meisenheimer complexes from non-su bstrate thiol analogues of glutathione offers new possibilities for ex amining the substrate interactions of glutathione transferases.