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

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 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 Sconjugates, 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 Sacetyldextran polymer derivatives ( M , 500000) 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 ihhibited the transferase activity in a dose-dependent fashion ; maximum inhibition of the enzyme activity was 40 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 Nacetyltransferase 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.

Determinants of the enzymatic activity and the subcellular localization of aspartate N-acetyltransferase

“…However, the determinants of its association with the ER and the orientation of its catalytic site (cytosolic or luminal) remain unknown. The related human enzyme, NAT8 (N-acetyltransferase 8), which shares approximately 30 % sequence identity with NAT8L and is expressed in kidneys and liver, was shown to catalyse the acetylation of cysteine-S-conjugates and to be also associated with the ER [11], consistent with the previous subcellular localization of this enzymatic activity [12]. No consensus has yet been reached on the subcellular localization of NAT8L, as Ariyannur et al [8] reported a partial mitochondrial localization of the endogenous protein in SH-SY5Y cells, using antibodies directed against NAT8L peptides.…”

Role of plasma albumin in renal elimination of a mercapturic acid

“…However, albuminligand interaction may become critically important if the rates of formation of such hazardous metabolites were increased, such as for bilirubin in acute hemolysis, or if animals were challenged with high doses of electrophilic xenobiotics, which would be metabolized to the corresponding mercapturic acids. Recent studies [20] revealed that the N-acetylation of a cysteine S-conjugate is catalyzed by the transferase at the cytoplasmic surface of endoplasmic reticulum in liver and kidney and that cytosolic organic anion-binding proteins (ligandins) facilitate this reaction by binding the N-acetylated reaction products and other competing organic anions. Thus, in addition to the interaction with cytosolic ligandins, binding to the circulating albumin may constitute an important step in the interorgan metabolism and transport leading to mercapturic acid biosynthesis and a final elimination of metabolites from the organism.…”

Hydrolysis of S-aryl-cysteinylglycine conjugates catalyzed by porcine kidney cortex membrane dipeptidase

“…Studies on microsomal preparations have shown that the active site of CCNAT is localized to the cytoplasmic side of the endoplasmic reticulum (ER) (Elce, 1970;Green and Elce, 1975;Okajima et al, 1984). The CCNAT co-substrate acetyl-CoA is localized in the cytosol at high concentrations to facilitate rapid and efficient N-acetylation of Cys conjugates (Garland et al, 1965).…”