Specificity of Aminoacylase III-Mediated Deacetylation of Mercapturic Acids

Abstract

ABSTRACT:Trichloroethylene (TCE) and other halogenated alkenes are known environmental contaminants with cytotoxic and nephrotoxic effects, and are potential carcinogens. Their metabolism via the mercapturate metabolic pathway was shown to lead to their detoxification. The final products of this pathway, mercapturic acids or N-acetyl-L-cysteine S-conjugates, are secreted into the lumen in the renal proximal tubule. The proximal tubule may also deacetylate mercapturic acids, and the resulting cysteine S-conjugates are transformed by cysteine S-conjugate ␤-lyases to nephrotoxic reactive thiols. The specificity and rate of mercapturic acid deacetylation may determine the toxicity of certain mercapturic acids; however, the exact enzymologic processes involved are not known in detail. In the present study we characterized the kinetics of the recently cloned mouse aminoacylase III (AAIII) toward a wide spectrum of halogenated mercapturic acids and N-acetylated amino acids. In general, the V max value of AAIII was significantly larger with chlorinated and brominated mercapturic acids, whereas fluorination significantly decreased it. The enzyme deacetylated mercapturic acids derived from the TCE metabolism including N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine (NA-1,2-DCVC) and N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine (NA-2,2-DCVC). Both mercapturic acids induced cytotoxicity in mouse proximal tubule mPCT cells expressing AAIII, which was decreased by an inhibitor of ␤-lyase, aminooxyacetate. The toxic effect of NA-2,2-DCVC was smaller than that of NA-1,2-DCVC, indicating that factors other than the intracellular activity of AAIII mediate the cytotoxicity of these mercapturic acids. Our results indicate that in proximal tubule cells, AAIII plays an important role in deacetylating several halogenated mercapturic acids, and this process may be involved in their cyto-and nephrotoxicity.Trichloroethylene (TCE) and other industrial solvents are shown to cause nephrotoxicity and hepatotoxicity, and are probable human carcinogens (Maltoni et al., 1988;Cummings and Lash, 2000). One of the TCE toxicity mechanisms involves the conjugation with glutathione, which is transformed by ␥-glutamyltransferase and dipeptidase to cysteine S-conjugates Berrnauer et al., 1996;Anders and Dekant, 1998). The formation of cysteine S-conjugates takes place mainly in liver and, to some extent, also in kidney. Cysteine S-conjugates may be acetylated in the liver and kidney into corresponding N-acetyl S-conjugates (mercapturic acids) and transported from the liver into the kidney. In the kidney proximal tubules, mercapturic acids may be secreted or deacetylated by aminoacylases into cysteine S-conjugates Dekant, 1994, 1998). The deacetylation reaction may play an important role in the renal nephrotoxicity since cysteine S-conjugates, but not mercapturates, are transformed by cysteine conjugate ␤-lyases into toxic metabolites (Hayden and Stevens, 1990;Commandeur et al., 1995). Cysteine S-conjugates also may be transformed by flavoprotein monooxygenase...

Structures of aminoacylase 3 in complex with acetylated substrates

“…Eight residues maintain van der Waals contacts with the substrate side chain (Asn70, Arg71, Ile127, Glu129, Tyr156, Phe164, Ser165, Cys175) where five of these are conserved between human, rat, and mouse AA2 and AA3 (Asn70, Arg71, His116, Ile127, and Phe281) (6). This would indicate that these residues are likely not responsible for specificity between AA3 and the substrate side chain of NAY.…”

“…The differences in loop dynamics could be attributed to the sequence divergence observed between the AA2 and AA3 loops (6).…”

“…Activity of wt-mAA3 and mutants was determined with NAY, NA-DCVC, and NAD by measuring the deacetylated product in a fluorescence assay in the presence of 0.1 mM CoCl 2 (6,35). The calibration curves were made with L-tyrosine, DCVC, and L-aspartate.…”

“…1) (1). There are three types of aminoacylases: (i) aminoacylase 1 (AA1) deacetylates neutral aliphatic N-acyl-α-amino acids and mercapturic acids; (ii) aminoacylase 2 or aspartoacylase (AA2) has a strict specificity for N α -acetyl-L-aspartate (NAD); and (iii) aminoacylase 3 (AA3) preferentially deacetylates N α -acetylated aromatic amino acids and mercapturic acids that are usually not deacetylated by AA1 (1)(2)(3)(4)(5)(6). Despite different substrate specificities, AA2 and AA3 have a high degree of sequence (42% identity) and structure homology but are both substantially different from AA1 (∼10% of sequence identity) (2,(6)(7)(8)(9)(10).…”

Kynurenine Aminotransferase III and Glutamine Transaminase L Are Identical Enzymes that have Cysteine S-Conjugate β-Lyase Activity and Can Transaminate l-Selenomethionine

“…The cysteine S-conjugate ␤-lyase reaction bypasses the normal detoxification of the cysteine S-conjugate to the mercapturate and generates nephrotoxic thiol metabolites (50 -54). Interestingly, not only are the cysteine S-conjugates of halogenated alkenes nephrotoxic, but even their precursor glutathione S-conjugates and corresponding mercapturates cause severe renal injury (55). The present work has extended the number of recognized mammalian cysteine S-conjugate ␤-lyases to include the KAT enzymes GTL/KAT III and AAT/KAT II.…”