Acylase I-catalyzed deacetylation of N-acetyl-L-cysteine and S-alkyl-N-acetyl-L-cysteines.
The aminoacylase that catalyzes the hydrolysis of N-acetyl-L-cysteine (NAC) was identified as acylase I after purification by column chromatography and electrophoretic analysis. Rat kidney cytosol was fractionated by ammonium sulfate precipitation, and the proteins were separated by ion-exchange column chromatography, gel-filtration column chromatography, and hydrophobic interaction column chromatography. Acylase activity with NAC and N-acetyl-L-methionine (NAM), a known substrate for acylase I, as substrates coeluted during all chromatographic steps. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the protein was purified to near homogeneity and had a subunit Mr of 43 000, which is identical with the Mr of acylase I from porcine kidney and bovine liver. n-Butylmalonic acid was a slow-binding inhibitor of acylase I and inhibited the deacetylation of NAC with a Ki of 192 +/- 27 microM. These results show that acylase I catalyzes the deacetylation of NAC. The acylase I-catalyzed deacetylation of a range of S-alkyl-N-acetyl-L-cysteines, their carbon and oxygen analogues, and the selenium analogue of NAM was also studied with porcine kidney acylase I. The specific activity of the acylase I-catalyzed deacetylation of these substrates was related to their calculated molar volumes and log P values. The S-alkyl-N-acetyl-L-cysteines with short (C0-C3) and unbranched S-alkyl substituents were good acylase I substrates, whereas the S-alkyl-N-acetyl-L-cysteines with long (>C3) and branched S-alkyl substituents were poLr acylase I substrates. The carbon and oxygen analogues of S-methyl-N-acetyl-L-cysteine and the carbon analogue of S-ethyl-N-acetyl-L-cysteine were poor acylase I substrates, whereas the selenium analogue of NAM was a good acylase I substrate.
Uttamsingh
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Department of Pharmacology and Physiology, University of Rochester, 601 Elmwood Avenue, Box 711, Rochester, New York 14642, USA.
Keller
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Anders
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MW
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry*; D000478:Alkylation; D000581:Amidohydrolases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002384:Catalysis; D003640:Dealkylation; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D013329:Structure-Activity Relationship; D013552:Swine
D000581:Amidohydrolases; C019672:aminoacylase I; D000111:Acetylcysteine