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 chromat ography and electrophoretic analysis. Rat kidney cytosol was fractiona ted by ammonium sulfate precipitation, and the proteins were separated by ion-exchange column chromatography, gel-filtration column chromato graphy, and hydrophobic interaction column chromatography. Acylase act ivity with NAC and N-acetyl-L-methionine (NAM), a known substrate for acylase I, as substrates coeluted during all chromatographic steps. So dium dodecyl sulfate-polyacrylamide gel electrophoresis showed that th e protein was purified to near homogeneity and had a subunit M-r of 43 000, which is identical with the M-r 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 K-i of 192 +/ - 27 mu M These results show that acylase I catalyzes the deacetylatio n of NAG. The acylase I-catalyzed deacetylation of a range of S-alkyl- N-acetyl-L-cysteines, their carbon and oxygen analogues, and the selen ium analogue of NAM was also studied with porcine kidney acylase I. Th e specific activity of the acylase I-catalyzed deacetylation of these substrates was related to their calculated molar volumes and lag P val ues. The S-alkyl-N-acetyl-L-cysteines with short (C-0-C-3) and unbranc hed S-alkyl substituents were good acylase I substrates, whereas the S -alkyl-N-acetyl-L-cysteines with long (>C-3) and branched S-alkyl subs tituents were poor acylase I substrates. The carbon and oxygen analogu es 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 seleni um analogue of NAM was a good acylase I substrate.