Topological aspects of microsomal N ‐acetyltransferase, an enzyme responsible for the acetylation of cysteine S ‐conjugates of xenobiotics

Acetylation of cysteine S ‐conjugates of xenobiotics by microsomal N ‐acetyltransferase is the final step of detoxicative metabolism leading to mercapturic acid biosynthesis. To elucidate the subcellular site of N ‐acetylation and the effective mechanism by which the final metabolites are eliminated from the organisms, topological aspects and catalytic properties of microsomal N ‐acetyltransferase and mercapturic acid biosynthesis in vivo were investigated. Intravenous administration of radioactive S ‐benzyl‐ L ‐cysteine, a model compound of cysteine S ‐conjugates, resulted in rapid acetylation of the conjugate in liver and kidney to a similar extent. The acetylation was followed by a rapid excretion of the metabolite, a mercapturic acid, into the urine; about 60% of the injected dose appeared in urine within 60 min of administration. Limited proteolysis of microsomal vesicles obtained from liver and kidney by chymotrypsin or trypsin inactivated the transferase by 49–62% and 62–73%, respectively. Proteolytic inactivation of the transferase was not significantly affected by the presence of 0.04% sodium deoxycholate by which the vesicles became permeable to macromolecules due to its detergent action. To determine the sidedness of the active site of N ‐acetyltransferase on the microsomal membranes, two S ‐acetyldextran polymer derivatives ( M t 5) of cysteine and N ‐acetylcysteine which represent an nonpermeant substrate and product for this enzyme, respectively, were examined for their effects on the vesicle‐associated enzyme activity. Both derivatives inhibited the transferase activity in a dose‐dependent fashion; maximum inhibition of the enzyme activity was 40% by the former and 60% by the latter. Sulfobromophthalein strongly inhibited the enzyme activity and, this inhibition was completely reversed by adding an equimolar amount of hepatic glutathione S ‐transferases (ligandins). In contrast to the strong inhibition by sulfobromophthalein itself, its glutathione S ‐conjugate did not inhibit the enzyme activity. These results indicate that the active site and the protease‐sensitive domain(s) of the microsomal N ‐acetyltransferase are localized on the outer surface (cytoplasmic side) of endoplasmic reticulum and that the ligandin(s) might protect membranous N ‐acetyltransferase‐from inhibition by organic anions by binding them and catalyzing the conjugation with glutathione.