Identification of N-acetylcysteine as a new substrate for rat liver microsomal glutathione transferase. A study of thiol ligands.

Abstract

N-Acetyl-L-cysteine serves as an efficient substrate for the rat liver microsomal glutathione transferase with 1-chloro-2,4-dinitrobenzene as second substrate (8.8 +/- 0.37 mumol/min mg). The activity is actually higher than that obtained with glutathione (2-4 mumol/min mg). In examining the activity of liver subcellular fractions, no activity with N-acetyl-L-Cys could be detected in dialyzed or N-ethylmaleimide-treated (in order to remove endogenous glutathione) cytosol. The activity in rat liver microsomes was 0.11 +/- 0.007 mumol/min mg, which is accounted 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 other second substrates than 1-chloro-2,4-dinitrobenzene (with varying electrophilicity) are used. The pH dependence of microsomal glutathione transferase was studied. The kcat/Km(1-chloro-2,4-dinitrobenzene) was dependent on pH with an apparent pKa of 6, > or = 9, and > or = 8 with saturating glutathione, gamma-L-Glu-L-Cys, and N-acetyl-L- cysteine, respectively. Apparently the enzyme has the ability to lower the pKa of glutathione by 3 orders of magnitude. The kcat/Km(thiol) did not vary appreciably with pH (except for N-acetyl-L-cysteine), indicating that no rate-determining deprotonation occurs on the enzyme itself between pH 5.5 and 9. The abilities of histidine-, lysine-, and arginine-selective reagents to inactivate the enzyme when N-acetyl-L-cysteine and gamma-L-Glu-L-Cys were used as substrates were investigated. The activity toward N-acetyl-L-cysteine was decreased considerably less after treatment with the arginine-selective reagent phenylglyoxal, as compared to the activity toward GSH and gamma-L-Glu-L-Cys. This indicates that an arginine makes contact with gamma-L-Glu residue in GSH. With the other reagent/substrate combinations tested the enzyme was inactivated almost completely. The ability of microsomal glutathione transferase to stabilize the Meisenheimer complex formation between 1,3,5-trinitrobenzene and various glutathione analogues, including non-substrate thiols, has been examined. It is shown that, in general, substrates exhibited higher formation constants (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-substrate thiol analogues of glutathione offers new possibilities for examining the substrate interactions of glutathione transferases.