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

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Abstract
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 were eliminated from the organisms, topological aspects and catalytic properties of microsomal N-acetyltransferase and mercapturic acid biosynthesis in vivo were investigated. I.v. administration of radioactive S-benzyl-L-cysteine, a model compound of cysteine S-conjugate, 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; .apprx. 60% of the injected dose appeared in urine within 60 min of administration. Limited proteolysis of microsomal vesicles obtained from rat 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, 2 S-acetyldextran polymer derivatives (MW 500,000) of cysteine and N-acetylcysteine which represented a 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. The active site and the protease-sensitive domain(s) of the microsomal N-acetyltransferase were localized on the outer surface (cytoplasmic side) of endoplasmic reticulum and the ligandin(s) might have protected membranous N-acetyltransferase from inhibition by organic anions by binding then and catalyzing the conjugation with glutathione.