Acylase I-Catalyzed Deacetylation of - American Chemical Society

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Chem. Res. Toxicol. 1998, 11, 800-809

Acylase I-Catalyzed Deacetylation of N-Acetyl-L-cysteine and S-Alkyl-N-acetyl-L-cysteines‡ Vinita Uttamsingh,† D. A. Keller,§ and M. W. Anders*,† Department of Pharmacology and Physiology, University of Rochester, 601 Elmwood Avenue, Box 711, Rochester, New York 14642, and Haskell Laboratory, P.O. Box 50, Elkton Road, Newark, Delaware 19714 Received January 26, 1998

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-Lmethionine (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 µM. 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-Lcysteines 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 poor acylase I substrates. The carbon and oxygen analogues of S-methylN-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.

Introduction NAC1 is an antioxidant with chemopreventive and therapeutic effects and is used clinically for the management of acetaminophen toxicity (1, 2) and congestive and obstructive lung disorders (3, 4). NAC may act directly as an antioxidant (5) or may serve as a L-cysteine prodrug and, thereby, support glutathione synthesis (1). Although NAC has been proposed for the therapy of HIV-1 infections (6), treatment of patients with AIDS with NAC failed to increase glutathione concentrations in plasma or peripheral blood mononuclear cells (7). NAC is deacetylated in isolated hepatocytes and supports glutathione synthesis in these cells (8). Also, human endothelial cells, rat lung, intestinal, and liver homogenates, and human liver homogenates catalyze the deacetylation of NAC (9, 10). The deacylation of N-acyl-L-amino acids is catalyzed by aminoacylases (for a review, see ref 11). Several ‡ A preliminary report of this work has appeared [Uttamsingh, V., and Anders, M. W. (1997) Aminoacylase I-catalyzed deacetylation of N-acetyl-L-cysteine. Fundam. Appl. Toxicol. 36 (Suppl., No. 1, Part 2), 79]. * Address correspondence to: M. W. Anders. Voice: 716-275-1681. Fax: 716-244-9283. E-mail: [email protected]. † University of Rochester. § Haskell Laboratory. 1 Abbreviations: NAC, N-acetyl-L-cysteine; NAM, N-acetyl-L-methionine; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

aminoacylases have been identified: acylase I (EC 3.5.1.14, N-acylamino acid hydrolase), aspartoacylase (EC 3.5.1.15, N-acetyl-L-aspartate amidohydrolase), acyllysine acylase (EC 3.5.17, N-acetyl-L-lysine amidohydrolase), and acylase III. Acylase I shows a preference for aliphatic N-acyl-R-amino acids. Aspartoacylase and acyllysine acylase are selective for N-acetyl-L-aspartic acid and Nacetyl-L-lysine, respectively. Acylase III shows a preference for N-acyl aromatic amino acids (12). Acylase I is well-characterized. Renal acylase I is a zinc-containing, homodimeric protein with a subunit Mr of 43 000 (13). Human and porcine cDNAs encoding the complete amino acid sequence of acylase I have been reported (14-16). Acylase III has not been studied in detail. A rat liver deacetylase with a substrate selectivity similar to that of acylase III has been reported (17). Acylases I and III may catalyze the observed hydrolysis of xenobiotic-derived mercapturates (S-substituted-Nacetyl-L-cysteines) in vivo in rabbits, rats, and guinea pigs and by rabbit, rat, and guinea pig kidney and liver tissue extracts (18). The mercapturates of several haloalkenederived, nephrotoxic, cytotoxic, and mutagenic cysteine S-conjugates are deacetylated in vivo in rats and by rat kidney cytosol and isolated renal proximal-tubular cells (19-21). The present experiments were designed to identify and characterize the aminoacylase that catalyzes the deacetylation of NAC and to study the effects of substrate

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Acylase I-Catalyzed Hydrolysis of N-Acetyl-L-cysteines

structure on acylase I activity. An enzyme purified from rat kidney, which was the rat ortholog of pig kidney acylase I, catalyzed the deacetylation of NAC and NAM. Studies on the effects of increasing both the chain length and the degree of branching of the S-alkyl substituent as well as replacement of sulfur with oxygen, carbon, or selenium showed that enzyme activity was related to the calculated molar volumes and log P values of the substrates. Finally, the acylase-catalyzed deacetylation of haloalkene-derived mercapturates of nephrotoxic, cytotoxic, and mutagenic cysteine S-conjugates was studied.

Materials and Methods Materials. N-Acetyl-L-cysteine, L-cysteine, N-acetyl-L-methionine, L-methionine, O-methyl-DL-serine, seleno-L-methionine, fluorescamine, 2,4,6-trinitrobenzenesulfonic acid, L-cysteine hydrochloride, and acylase I (grade III) were purchased from Sigma Chemical Co. (St. Louis, MO). n-Butylmalonic acid (pentane-1,1-dicarboxylic acid), methyl iodide, ethyl iodide, propyl iodide, isopropyl iodide, n-butyl iodide, isobutyl iodide, 2-methyl-2-propanol, and 2-aminopentanoic acid (L-norvaline) were obtained from Aldrich Chemical Co. (Milwaukee, WI). N-Acetyl-L-2-aminohexanoic acid (N-acetyl-L-norleucine) was obtained from Indofine Chemical Co., Inc. (Somerville, NJ). Analytical Methods. Melting points were determined with a Mel-Temp melting point apparatus and are uncorrected. 1H NMR spectra were recorded with a Bruker 270 MHz spectrometer operating at 270-MHz for 1H. Chemical shifts, δ, are reported in parts per million (ppm). The HOD resonance at 4.7 ppm was used as the internal standard for 1H NMR spectra when D2O was the solvent. The solvent resonance peak at 2.47 ppm was used as the internal standard for 1H NMR spectra when dimethyl sulfoxide-d6 was the solvent. Elemental analyses were determined by Midwest Microlab (Indianapolis, IN). Syntheses. S-Alkyl-N-acetyl-L-cysteines. S-Methyl-, Sethyl-, S-n-propyl-, S-isopropyl-, S-n-butyl-, and S-isobutyl-Nacetyl-L-cysteines were synthesized as described by Grenby and Young (22). The compounds were purified as described below. S-Methyl-N-acetyl-L-cysteine. The reaction mixture was brought to pH 1 with concentrated HCl and extracted with ethyl acetate. The ethyl acetate layer was separated, and the solvent was removed in vacuo. The crude product was purified by silica gel column chromatography. The column was eluted with 1% methanol-ethyl acetate containing 0.01% acetic acid, and the product was recrystallized from ethyl acetate-petroleum ether. Pure white crystals were obtained: mp 78-79 °C [lit. mp 82 °C (23)]; 1H NMR (D2O) δ 2.0 (s, 3H, NHCOCH3), 2.1 (s, 3H, CH2SCH3), 2.8-3.1 (m, 2H, CHCH2SCH3), 4.5-4.6 (m, 1H, (COOH)CH(NHCOCH3)). S-Ethyl-N-acetyl-L-cysteine. The reaction mixture was brought to pH 1 with concentrated HCl and extracted with ethyl acetate. The ethyl acetate layer was separated, and the solvent was removed in vacuo. The crude product was purified by silica gel column chromatography. The column was eluted with 1% methanol-ethyl acetate containing 0.01% acetic acid, and the product was recrystallized from ethyl acetate-hexane. Pure white crystals were obtained: mp 78-79 °C [lit. mp 78.5-79.5 °C (24)]; 1H NMR (D2O) δ 1.2 (t, 3H, CH2SCH2CH3), 2.0 (s, 3H, NHCOCH3), 2.5 (q, 2H, CH2SCH2CH3), 2.8-3.1 (m, 2H, CHCH2SCH2CH3), 4.5-4.6 (m, 1H, (COOH)CH(NHCOCH3)). S-Propyl-N-acetyl-L-cysteine. The reaction mixture was brought to pH 1 with concentrated HCl and extracted with ethyl acetate. The ethyl acetate layer was separated, and the solvent was removed in vacuo. The crude product was purified by silica gel column chromatography. The column was eluted with 1% methanol-ethyl acetate containing 0.01% acetic acid, and the product was recrystallized from water. Pure white crystals were obtained: mp 96 °C [lit. mp 96 °C (22)]; 1H NMR (D2O) δ 0.9 (t, 3H, SCH2CH2CH3), 1.5 (m, 2H, SCH2CH2CH3), 2.5 (t, 2H, SCH2-

Chem. Res. Toxicol., Vol. 11, No. 7, 1998 801 CH2CH3), 2.0 (s, 1H, NHCOCH3), 2.8-3.1 (m, 2H, CHCH2S), 4.5 (m, 1H, (COOH)CH(NHCOCH3)). S-Isopropyl-N-acetyl-L-cysteine. The reaction mixture was brought to pH 1 with concentrated HCl and extracted with ethyl acetate. The ethyl acetate layer was separated, and the solvent was removed in vacuo. The crude product was purified by recrystallization from water. Pure white crystals were obtained: mp 130 °C [lit. mp 134 °C (25)]; 1H NMR (D2O) δ 1.2 (d of d, 6H, -CH(CH3)2), 2.0 (s, 3H, NHCOCH3), 3.0 (m, 3H, CH2-S-CH), 4.5 (m, 1, (COOH)CH(NHCOCH3)). S-n-Butyl-N-acetyl-L-cysteine. The reaction mixture was brought to pH 1 with concentrated HCl and extracted with ethyl acetate. The ethyl acetate layer was separated, and the solvent was removed in vacuo. The crude product was purified by silica gel column chromatography. The column was eluted with 1% methanol-ethyl acetate containing 0.01% acetic acid, and the product was recrystallized from ethyl acetate-hexane. Pure white crystals were obtained: mp 67-69 °C [lit. mp 67 °C (26)]; 1H NMR (D O) δ 0.8 (t, 3H, SCH CH CH CH ), 1.2-1.4 (m, 2H, 2 2 2 2 3 SCH2CH2CH2CH3), 1.4-1.6 (m, 2H, SCH2CH2CH2CH3), 2.5 (t, 2H, SCH2CH2CH2CH3), 2.0 (s, 3H, NHCOCH3), 2.8-3.1 (m, 2H, CHCH2S), 4.5 (m, 1H, (COOH)CH(NHCOCH3)). S-Isobutyl-N-acetyl-L-cysteine. The reaction mixture was brought to pH 1 with concentrated HCl and extracted with ethyl acetate. The ethyl acetate layer was separated, and the solvent was removed in vacuo. The crude product was purified by recrystallization from water. Pure white crystals were obtained: mp 134 °C; 1H NMR (D2O) for the new compound δ 0.9 (d, 6H, CH(CH3)2), 1.4-1.6 (m, CH2CH(CH3)2), 2.0 (s, 3H, NHCOCH3), 2.4 (d, 2H, CH2SCH2CH(CH3)2), 2.8-3.1 (m, 2H, CHCH2SCH2(CH3)2), 4.5 (m, 1H, (COOH)CH(NHCOCH3)). Elemental Anal. Calcd for C9H17NO3S: C, 49.32; H, 7.76; N, 6.39. Found: C, 49.46; H, 7.89; N, 6.57. S-tert-Butyl-N-acetyl-L-cysteine. S-tert-Butyl-N-acetyl-Lcysteine was obtained by the acetylation of S-tert-butyl-Lcysteine hydrochloride. S-tert-Butyl-L-cysteine hydrochloride was synthesized as described by Pastuszak and Chimiak (27) and then acetylated with acetyl chloride/triethylamine (28). The reaction mixture was stirred overnight and filtered, and the solvent was evaporated. The solid product obtained was dissolved in ethyl acetate, and the solution was filtered. The ethyl acetate was evaporated in vacuo, and the crude product obtained was purified by silica gel column chromatography. The column was eluted with 0.5% methanol-ethyl acetate containing 0.01% acetic acid, and the product was recrystallized from methanolwater. Pure white crystals were obtained: mp 130 °C dec; 1H NMR (DMSO-d6) for the new compound δ 1.4 (s, 9H, C(CH3)3), 2.0 (s, 3H, NHCOCH3), 2.8-3.0 (m, 2H, CHCH2S), 4.4 (m, 1H, (COOH)CH(NHCOCH3)), 8.2-8.4 (d, 1H, NHCOCH3). Elemental Anal. Calcd for C9H17NO3S: C, 49.32; H, 7.76; N, 6.39. Found: C, 49.75; H, 8.00; N, 6.72. N-Acetyl-L-2-aminopentanoic Acid, N-Acetyl-O-methylDL-serine, and N-Acetylseleno-L-methionine. These were obtained by the acetylation of L-2-aminopentanoic acid, Omethyl-DL-serine, and seleno-L-methionine, respectively, with pentafluorophenyl acetate in DMF (29). The N-acetylated compounds were obtained as oily residues from the reaction mixtures after evaporation of the solvent in vacuo. The crude products were solidified by repeatedly adding hexane and evaporating in a rotary evaporator. The solid products obtained were purified by recrystallization from ethyl acetate-hexane. N-Acetyl-L-2-aminopentanoic Acid. The product gave white crystals: mp 97-99 °C [lit. mp 100 °C (30)]; 1H NMR (D2O) δ 0.75 (t, 3H, CHCH2CH2CH3), 1.25-1.4 (m, 2H, CHCH2CH2CH3), 1.6-1.8 (m, 2H, CHCH2CH2CH3), 2.0 (s, 3H, NHCOCH3), 4.2 (m, 1H, (COOH)CH(NHCOCH3)). O-Methyl-N-acetyl-DL-serine. The product gave a yellowish-white powder: mp 105-107 °C [lit. mp 108-109 °C (31)]; 1H NMR (D O) δ 2.0 (s, 3H, NHCOCH ), 3.3 (s, 3H, CH OCH ), 2 3 2 3 3.65-3.9 (m, 2H, CH2OCH3), 4.5 (m, 1H, (COOH)CH(NHCOCH3)).

802 Chem. Res. Toxicol., Vol. 11, No. 7, 1998 N-Acetylseleno-L-methionine. The product was obtained as a white powder: mp 107 °C; 1H NMR (D2O) for the new compound δ 1.9 (s, 3H, SeCH3), 2.0 (s, 3H, NHCOCH3), 2.452.7 (m, 4H, CHCH2CH2Se), 4.4-4.5 (m, 1H, (COOH)CH(NHCOCH3)). Elemental Anal. Calcd for C7H13NO3Se: C, 35.30; H, 5.46; N, 5.88. Found: C, 35.41; H, 5.58; N, 5.83. S-(1,1,2,2-Tetrafluoroethyl)-N-acetyl-L-cysteine and S-(2Bromo-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine. These were prepared by the acetylation of S-(1,1,2,2-tetrafluoroethyl)-Lcysteine (32) and S-(2-bromo-1,1,2-trifluoroethyl)-L-cysteine (33), respectively, with pentafluorophenyl acetate in DMF, as described above. The acetylated products were purified by silica gel column chromatography. The column was eluted with 50% methylene chloride-ethyl acetate, and the product was recrystallized from ethyl acetate-hexane. S-(1,1,2,2-Tetrafluoroethyl)-N-acetyl-L-cysteine was obtained as a white powder; its physical constants were identical with the reported values (32). S-(2-Bromo-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine was obtained as a white powder: mp 103-104 °C; 1H NMR (D2O) for the new compound δ 2.0 (s, 3H, NHCOCH3), 3.2-3.5 (m, 2H, CHCH2SCF2), 4.5-4.6 (m, 1H, (COOH)CH(NHCOCH3)), 6.76.8, 6.9-7.0 (d of t, 1H, CH2SCHF2CHBrF). Elemental Anal. Calcd for C7H8NO3SBrF3: C, 26.01; H, 2.48; N, 4.33. Found: C, 26.40; H, 2.88; N, 4.26. S-(2-Chloro-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine. This was prepared as described by Boogaard et al. (20). Enzyme Purification. Kidneys from male Sprague-Dawley rats (Pel-freeze, Rogers, AR) were homogenized in 50 mM potassium phosphate buffer (pH 7.4) at 4 °C. All purification steps were performed at 4 °C. The homogenate was centrifuged at 9000g, and the supernatant was centrifuged at 100000g to obtain the cytosolic fraction. Solid ammonium sulfate was added to the cytosol to 50% saturation, and the mixture was stirred for 1 h and then centrifuged at 9000g for 25 min. The precipitated proteins were dissolved in a minimum volume of 10 mM potassium phosphate buffer (pH 7.4) and dialyzed against the same buffer for 24 h. The dialyzed protein solution was concentrated to 32 mL in an Amicon ultrafiltration cell and applied to a DEAE-cellulose column (5.0 × 32 cm; Express IonD, Whatman, Hillsboro, OR) equilibrated with 10 mM potassium phosphate buffer (pH 7.4). The column was eluted with 700 mL of 10 mM potassium phosphate buffer (pH 7.4) and then with a linear gradient of 10-200 mM potassium phosphate buffer (pH 7.4). Eluate fractions (8.2 mL) were collected and assayed for protein concentrations and enzyme activities. Fractions with the highest activity were combined and concentrated in an Amicon ultrafiltration cell, and 42 mL of the concentrated fraction was applied to a Sephacryl S-200 HR (5.0 × 40 cm; Pharmacia, Piscataway, NJ) column equilibrated with 10 mM potassium phosphate buffer (pH 7.4); the column was eluted with the same buffer. Eluate fractions (4 mL) were collected and assayed for protein concentrations and enzyme activities. Fractions with the highest activities were combined and concentrated in an Amicon ultrafiltration cell, and 5 mL of the concentrated fraction was applied to a Phenyl-Sepharose column [1.5 × 8 cm; Phenyl Sepharose 6 fast flow (low substitution), Pharmacia, Piscataway, NJ] equilibrated with 50 mM potassium phosphate buffer (pH 7.0) containing 1.7 M (NH4)2SO4. Proteins were eluted first with three column volumes of the column equilibration buffer and then with three column volumes of 50 mM potassium phosphate buffer (pH 7.0). Eluate fractions (1 mL) were collected and assayed for protein concentrations and enzyme activities and were analyzed by SDS-PAGE (10% acrylamide). Proteins were visualized by Coomassie blue staining. The purified acylase was stored at 4 °C for 1 month without significant loss of acylase activity. Protein concentrations were determined by the method of Bradford (34) with bovine serum albumin as the standard. Activity Assays. Acylase I activity was determined by measuring the formation of deacetylated L-amino acids. LMethionine formed by the deacetylation of NAM was determined by reaction with fluorescamine (35). The reaction mixture (1

Uttamsingh et al. mL) contained 0.1-0.5 mL of protein solution and 2 µmol of substrate in 50 mM potassium phosphate buffer (pH 7.4). The reaction mixture was incubated at 37 °C for 1 h, and the reaction was stopped by the addition of 0.2 mL of 20% trichloroacetic acid. The mixture was allowed to stand for 10 min in an ice bath and then centrifuged (500g, 10 min). A sample (40 µL) of the supernatant was added to 3.6 mL of 50 mM potassium phosphate buffer (pH 7.4), and the volume was brought to 4 mL by the addition of water. Fluorescamine (300 µL of a solution containing 10 mg of fluorescamine dissolved in 33 mL of acetone) was added to the sample, and the fluorescence intensity (390-nm excitation, 475-nm emission) was measured immediately with a Perkin-Elmer LS-5 fluorescence spectrophotometer (Norwalk, CT). A standard curve was prepared with L-methionine. L-Cysteine formed by the deacetylation of NAC was determined by reaction with 2,4,6-trinitrobenzenesulfonic acid (36). (The thiol groups in L-cysteine and NAC interfered with the fluorescamine assay.) The deacetylation reaction was performed as described for NAM. A sample (200 µL) of the supernatant was added to 2.8 mL of 50 mM potassium phosphate buffer (pH 7.4). 2,4,6-Trinitrobenzenesulfonic acid (0.5 mL of a 0.07% solution in the assay buffer) was added to the sample, and the mixture was incubated at 50 °C for 30 min. The reaction mixture was cooled to room temperature, and the absorbance at 420 nm was measured with a Perkin-Elmer Lambda 3A spectrophotometer (Norwalk, CT). Acylase I activity with NAC, NAM, S-methyl-, S-ethyl-, S-npropyl-, S-isopropyl-, S-n-butyl-, S-isobutyl-, and S-tert-butylN-acetyl-L-cysteines, N-acetyl-L-2-aminopentanoic acid, N-acetylL-2-aminohexanoic acid, N-acetylseleno-L-methionine, O-methylN-acetyl-DL-serine, S-(1,1,2,2-tetrafluoroethyl)-N-acetyl-L-cysteine, S-(2-chloro-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine, and S-(2bromo-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine was determined with commercially available porcine kidney acylase I. The reaction mixtures contained 3.5 units of acylase I and 4 µmol of substrate in a final volume of 1 mL of 50 mM potassium phosphate buffer (pH 7.4). The reaction mixtures containing NAC, NAM, S-methyl-, S-ethyl-, and S-n-propyl-N-acetyl-Lcysteines, N-acetylseleno-L-methionine, S-(1,1,2,2-tetrafluoroethyl)-N-acetyl-L-cysteine, S-(2-chloro-1,1,2-trifluoroethyl)-Nacetyl-L-cysteine, and S-(2-bromo-1,1,2-trifluoroethyl)-N-acetylL-cysteine were incubated for 30 min at 37 °C, and the reaction mixtures containing S-isopropyl-, S-n-butyl-, S-isobutyl-, and S-tert-butyl-N-acetyl-L-cysteines, N-acetyl-L-2-aminopentanoic acid, N-acetyl-L-2-aminohexanoic acid, and O-methyl-N-acetylDL-serine were incubated for 1 h at 37 °C. The amounts of L-amino acids formed were measured as described above. Kinetic Analyses. The kinetics of the deacetylation of NAC, NAM, S-methyl-, S-ethyl-, and S-n-propyl-N-acetyl-L-cysteines, and N-acetylseleno-L-methionine was studied with commercially available porcine kidney acylase I. For the determination of the Km and Vmax, the reaction mixtures contained 3.5 units of acylase I and 1.0-16.0 mM substrate in a final volume of 1 mL of 50 mM potassium phosphate buffer (pH 7.4). The reaction mixtures were incubated at 37 °C for 10 min, and the amounts of L-amino acids formed were measured as described above. Experiments were performed in triplicate. The Km and Vmax were computed by fitting the data to the Michaelis-Menten equation with the EZ-FIT program (Perrella Scientific, Inc., Conyers, GA). The inhibition of the acylase I-catalyzed deacetylation of NAC by n-butylmalonic acid was also studied. The reaction mixtures contained 53 units of pig kidney acylase I, 2 mM NAC, and 0, 0.2, 0.4, or 0.6 mM n-butylmalonic acid in 5 mL of 50 mM potassium phosphate buffer (pH 7.4); reactions were started by addition of enzyme. The reaction mixtures were incubated at 37 °C, and 50-µL samples were withdrawn at 3-min intervals and added to 2.95 mL of 50 mM potassium phosphate buffer (pH 7.4). The amount of L-cysteine formed was quantified as described above. Experiments were performed in triplicate. Ki values were determined from the progress curves as described


Chem. Res. Toxicol., Vol. 11, No. 7, 1998 803

by Morrison et al. (37). The progress curves were fitted to a one-step mechanism for reversible, slow-binding inhibition, which is described by the equation: P ) Vst + (Vo - Vs)(1 e-kt)/k. Kinetic constants were computed by the EZ-FIT program. The inhibition of the acylase I-catalyzed deacetylation of NAM by the sulfur-lacking analogue N-acetyl-L-2-aminohexanoic acid was also studied. The reaction mixtures (1 mL) contained NAM (1-10 mM), 3.5 units of pig kidney acylase I, and 1, 2, 4, or 6 mM N-acetyl-L-2-aminohexanoic acid in 50 mM potassium phosphate buffer (pH 7.4) and were incubated at 37 °C for 20 min. The amounts of L-methionine formed were measured as described earlier. Experiments were performed in triplicate. The data were analyzed by the EZ-FIT program. Calculations. The log P and molar refractivity (CMR) were calculated as described by Hansch and Leo (38). The molar volumes were calculated as molar refractivity, which is a sum of the atom or bond refractivities, and considered a form of molar volume “corrected” for double bonds. The units for CMR are cm3 × 0.1. Version 3.55 of the CLOGP program and version 3.4 of the Molar Refractivity Calculator from BioByte (Pomona, CA) were used for the calculations.

Results Enzyme Purification. Rat kidney cytosol was fractionated by ammonium sulfate precipitation, DEAEcellulose column chromatography, Sephacryl S-200 column chromatography, and Phenyl-Sepharose column chromatography. The proteins obtained by ammonium sulfate fractionation of rat kidney cytosol were applied to a DEAE-cellulose column, which was eluted with 10 mM potassium phosphate buffer (pH 7.4) (Figure 1A). Fractions 134-206, which had high activity with NAC and NAM as substrates, were concentrated and applied to a Sephacryl S-200 column. Acylase activity with NAC and NAM as substrates eluted from the Sephacryl S-200 column as a single peak in fractions 86-102 (Figure 1B). The acylase-containing fractions from the Sephacryl S-200 column were applied to a Phenyl-Sepharose column. The acylase in fractions 22 and 23 from the PhenylSepharose column showed a purification of 200- and 289fold with NAC and NAM, respectively, as the substrates (Figure 1C) (Table 1). Fractions from the PhenylSepharose column were analyzed by SDS-PAGE, which showed that the protein was purified to near homogeneity and had a Mr of 43 000 (Figure 2). In addition, the purified protein and commercial acylase I had identical Mr values when analyzed by SDS-PAGE. The Km and Vmax for the rat kidney acylase I-catalyzed deacetylation of NAC were 4.4 mM and 44.7 µmol/mg of protein/min, respectively. n-Butylmalonic acid inhibited the pig kidney acylase I-catalyzed deacetylation of NAC with a Ki of 192 ( 27 µM. The progress curves showed that n-butylmalonic was a slow-binding inhibitor of the acylase I-catalyzed deacetylation of NAC (Figure 3). The koff and kon values could not be satisfactorily determined because of large coefficients of variation. Structure-Activity Studies. The acylase I-catalyzed deacetylation of NAC, a range of S-alkyl-N-acetylL-cysteines and their carbon and oxygen analogues, NAM, and the selenium analogue of NAM was studied. The effects of increasing the side-chain length and degree of branching of S-alkyl substituents on acylase I specific activity were correlated with the calculated molar volumes and log P values. The kinetic data and calculated molar volumes and log P values of the substrates studied are presented in Table 2. The acylase I specific activity

Figure 1. Purification of rat kidney aminoacylase I by column chromatography. (A) DEAE-cellulose column chromatography of 0-50% ammonium sulfate fraction from rat kidney cytosol. The column was eluted with 10 mM potassium phosphate buffer (pH 7.4) and then with a linear gradient of 10-200 mM potassium phosphate buffer (pH 7.4). Eluate fractions (8.2 mL) were collected and assayed for protein concentrations (9), and enzyme activities with NAC (O) and NAM ([) as substrates were measured as described in Materials and Methods. Fractions 134-206 had high aminoacylase activity. (B) Sephacryl S-200 HR column chromatography of fractions 134-206 obtained from DEAE-cellulose column chromatography. The column was eluted with 10 mM potassium phosphate buffer (pH 7.4). Eluate fractions (4 mL) were collected and assayed for protein concentrations (9), and enzyme activities with NAC (O) and NAM ([) as substrates were measured as described in Materials and Methods. Fractions 86-102 had high aminoacylase activity. (C) Phenyl-Sepharose column chromatography of fractions 86-102 obtained from Sephacryl S-200 HR column chromatography. The column was eluted with 50 mM potassium phosphate buffer (pH 7.0) containing 1.7 M (NH4)2SO4 (fractions 0-10) and then with 50 mM potassium phosphate buffer (pH 7.0) (fractions 11-25). Eluate fractions (1 mL) were collected and assayed for protein concentrations (9), and enzyme activities with NAC (O) and NAM ([) as substrates were measured as described in Materials and Methods. Fractions 22 and 23 had high aminoacylase activity.

with NAC and the S-alkyl-N-acetyl-L-cysteines as substrates followed the order: NAC < S-methyl-N-acetyl-L-

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Uttamsingh et al.

Table 1. Purification of Aminoacylase from Rat Kidney N-Acetyl-L-cysteine

N-Acetyl-L-methionine

fraction

vol (mL)

protein concn (mg/mL)

total protein (mg)

specific activity (nmol/mg/min)

total activity (nmol/min)

purification (fold)

specific activity (nmol/mg/min)

total activity (nmol/min)

purification (fold)

cytosol 0-50% (NH4)2SO4 DEAE-cellulose Sephacryl S-200 Phenyl-Sepharose

215 32 45 50 24

45.4 16.5 4.2 1.0 0.009

9675 528 189 50 0.22

60 180 343 1377 12000

580500 95040 64827 68850 2640

3.0 5.7 23 200

45 160 337 1117 13000

435375 84480 63693 55850 2860

3.6 7.5 25.0 289.0

Figure 2. Electrophoretic analysis of purified rat kidney acylase I. Fractions from the Phenyl-Sepharose column chromatography step (see Figure 1C) were analyzed by SDS-PAGE, as described in Materials and methods. Fractions were 3-8 and 19-23. Fractions 22 and 23 and commercial porcine kidney acylase I (lane AI) had identical Mr.

Figure 3. Inhibition of the acylase I-catalyzed deacetylation of NAC by n-butylmalonate. The reaction mixtures contained 53 units of pig kidney acylase I, 2 mM NAC, and 0 (b), 0.2 (9), 0.4 (2), or 0.6 ([) mM n-butylmalonate in 5 mL of 50 mM potassium phosphate buffer (pH 7.4) and were incubated at 37 °C. Enzyme activity was measured as described in Materials and Methods.

cysteine < S-ethyl-N-acetyl-L-cysteine > S-n-propyl-Nacetyl-L-cysteine > S-n-butyl-N-acetyl-L-cysteine > S-isopropyl-N-acetyl-L-cysteine ≈ S-isobutyl-N-acetyl-Lcysteine. No deacetylation of S-tert-butyl-N-acetyl-Lcysteine was detected. The acylase I specific activities with NAM and S-ethyl-N-acetyl-L-cysteine were similar. Acylase I activity with N-acetyl-L-2-aminopentanoic acid and O-methyl-N-acetyl-DL-serine, the carbon and

oxygen analogues of S-methyl-N-acetyl-L-cysteine, was lower than with S-methyl-N-acetyl-L-cysteine. No deacetylation of the N-acetyl-L-2-aminohexanoic acid, the carbon analogue of S-ethyl-N-acetyl-L-cysteine, was detected. Furthermore, N-acetyl-L-2-aminohexanoic acid inhibited the acylase I-catalyzed deacetylation of NAM with a Ki of 2.02 ( 0.33 mM. The analysis of the inhibition data showed that N-acetyl-L-2-aminohexanoic acid was a competitive inhibitor of the acylase I-catalyzed deacetylation of NAM (data not shown). Also, acylase I activity with N-acetylseleno-L-methionine, the selenium analogue of NAM, was lower than with NAM. A plot of acylase I specific activity against calculated molar volumes (Figure 4a) showed that the hydrolysis of S-alkyl-N-acetyl-L-cysteines with straight-chain Salkyl substituents increased with increasing molar volume (NAC < S-methyl-N-acetyl-L-cysteine < S-ethyl-Nacetyl-L-cysteine) and then decreased with further increases in molar volumes (S-n-propyl-N-acetyl-L-cysteine > S-n-butyl-N-acetyl-L-cysteine). For the S-alkylN-acetyl-L-cysteines with branched-chain S-alkyl substituents, acylase I activity decreased with increases in the degree of branching of the side chain, even though the substrates with branched-chain S-alkyl substituents had identical calculated molar volumes as their straightchain analogues. Thus, S-n-propyl-N-acetyl-L-cysteine and S-isopropyl-N-acetyl-L-cysteine had identical calculated molar volumes, but acylase I activity with Sisopropyl-N-acetyl-L-cysteine was lower than activity with S-n-propyl-N-acetyl-L-cysteine. Similarly, S-n-butyl-N-acetyl-L-cysteine, S-isobutyl-N-acetyl-L-cysteine, and S-tert-butyl-N-acetyl-L-cysteine had identical calculated molar volumes, but acylase I activity with S-isobutyl-Nacetyl-L-cysteine was lower than with S-n-butyl-N-acetylL-cysteine; activity with S-tert-butyl-N-acetyl-L-cysteine was not detected. Acylase I activity with O-methyl-Nacetyl-DL-serine, N-acetyl-L-2-aminopentanoic acid, and N-acetyl-L-2-aminohexanoic acid was lower than with sulfur-containing analogues even though their calculated molar volumes were lower. A plot of acylase I specific activity against calculated log P values showed a trend similar to that observed with calculated molar volumes of S-alkyl-N-acetyl-L-cysteines with unbranched S-alkyl substituents (Figure 5a). SIsopropyl-N-acetyl-L-cysteine, S-isobutyl-N-acetyl-L-cysteine, and S-tert-butyl-N-acetyl-L-cysteine had lower log P values than their corresponding unbranched analogues, but their rates of deacetylation were also lower. Acylase I activity with O-methyl-N-acetyl-DL-serine and N-acetylL-2-aminopentanoic acid was lower than with the sulfurcontaining analogue even though their calculated log P values were lower and higher, respectively. Also, acylase I activity with N-acetyl-L-2-aminohexanoic acid was lower than with sulfur-containing analogue even though its calculated log P value was higher.



Table 2. Relationship between Molar Volume or log P and Kinetics of S-Substituted-N-acetyl-L-cytseines

substrate

molar volume

log P

specific activitya (µmol/mg of protein/min)

N-acetyl-L-cysteine S-methyl-N-acetyl-L-cysteine N-acetyl-L-2-aminopentanoic acid S-ethyl-N-acetyl-L-cysteine N-acetyl-L-2-aminohexanoic acid N-acetyl-L-methionine S-n-propyl-N-acetyl-L-cysteine S-isopropyl-N-acetyl-L-cysteine S-n-butyl-N-acetyl-L-cysteine S-isobutyl-N-acetyl-L-cysteine S-tert-butyl-N-acetyl-L-cysteine O-methyl-N-acetyl-DL-serine N-acetylseleno-L-methionine S-(1,1,2,2-tetrafluoroethyl)-N-acetyl-L-cysteine S-(2-chloro-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine S-(2-bromo-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine

3.90 4.36 4.02 4.82 4.48 4.82 5.29 5.29 5.75 5.75 5.75 3.71 5.12 4.89 5.36 5.64

-0.642 -0.301 0.436 0.228 0.965 -0.487 0.757 0.537 1.286 1.066 0.936 -1.063 not available 1.560 1.900 2.040

19.9 ( 3.2 27.1 ( 4.7 3.1 ( 0.5 33.1 ( 3.1 activity not detected 30.8 ( 3.0 24.7 ( 1.2 1.8 ( 1.3 5.1 ( 1.5 1.5 ( 1.1 activity not detected 4.7 ( 0.5b 14.9 ( 1.9 161 ( 11.3 68 ( 2.3 51.6 ( 5.2

Km Vmax Vmax/Km (mM) (µmol/mg/min) (mL/mg/min) 4.4 2.1 >10.0 3.4 >10.0 2.1 0.8 >10.0 3.7 >10.0 >10.0 >10.0 4.0 ND ND ND

44.7 70.9 NDc 95.2 ND 87.1 45.5 ND 18.5 ND ND ND 58.4 ND ND ND

10.2 33.8 28.0 41.5 56.9 5.0

14.6

a Substrate concentration ) 4 mM. b Substrate concentration ) 8 mM. c ND, not determined. Data shown in the table were obtained with the commercially available porcine kidney acylase I.

Figure 4. (a) Relationship between specific activity and calculated molar volumes of NAC, NAM, S-alkyl-N-acetyl-L-cysteines, O-methyl-N-acetyl-L-serine, N-acetyl-L-2-aminopentanoic acid, N-acetyl-L-2-aminohexanoic acid, and N-acetylseleno-L-methionine. (Note: Y-axis range is 0-40 units.) (b) Relationship between specific activity and calculated molar volumes of S-(1,1,2,2tetrafluoroethyl)-N-acetyl-L-cysteine, S-(2-chloro-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine, and S-(2-bromo-1,1,2-trifluoroethyl)-N-acetylL-cysteine. (Note: Y-axis range is 40-180 units.) The reaction mixtures contained 3.5 units of pig kidney acylase I and 4 mM substrate in 1 mL of 50 mM potassium phosphate buffer (pH 7.4) and were incubated at 37 °C for 30 min or 1 h. The reaction mixtures containing NAC, NAM, S-methyl-, S-ethyl-, and S-n-propyl-N-acetyl-L-cysteines, N-acetylseleno-L-methionine, S-(1,1,2,2-tetrafluoroethyl)-N-acetyl-L-cysteine, S-(2-chloro-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine, and S-(2-bromo-1,1,2-trifluoroethyl)-N-acetyl-Lcysteine were incubated for 30 min, whereas the reaction mixtures containing S-isopropyl-, S-n-butyl-, S-isobutyl-, and S-tert-butylN-acetyl-L-cysteines, N-acetyl-L-2-aminopentanoic acid, N-acetyl-L-2-aminohexanoic acid, and O-methyl-N-acetyl-DL-serine were incubated for 1 h. Enzyme activity was measured as described in Materials and Methods.


Uttamsingh et al.

Figure 5. (a) Relationship between specific activity and calculated log P values of NAC, NAM, S-alkyl-N-acetyl-L-cysteines, O-methylN-acetyl-L-serine, N-acetyl-L-2-aminopentanoic acid, and N-acetyl-L-2-aminohexanoic acid. (Note: Y-axis range is 0-40 units.) (b) Relationship between specific activity and calculated log P values of S-(1,1,2,2-tetrafluoroethyl)-N-acetyl-L-cysteine, S-(2-chloro1,1,2-trifluoroethyl)-N-acetyl-L-cysteine, and S-(2-bromo-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine. (Note: Y-axis range is 40-180 units.) The reaction mixtures contained 3.5 units of pig kidney acylase I and 4 mM substrate in 1 mL of 50 mM potassium phosphate buffer (pH 7.4) and were incubated at 37 °C for 30 min or 1 h. The reaction mixtures containing NAC, NAM, and S-methyl-, S-ethyl-, and S-n-propyl-N-acetyl-L-cysteines were incubated for 30 min, whereas the reaction mixtures containing S-isopropyl-, S-n-butyl-, S-isobutyl-, and S-tert-butyl-N-acetyl-L-cysteines, N-acetyl-L-2-aminopentanoic acid, N-acetyl-L-2-aminohexanoic acid, and O-methylN-acetyl-DL-serine were incubated for 1 h. Enzyme activity was measured as described in Materials and Methods.

The relationship between Vmax/Km and calculated molar volumes and between Vmax/Km and log P values is shown in Table 2. S-n-Propyl-N-acetyl-L-cysteine exhibited the highest Vmax/Km. Acylase I-Catalyzed Deacetylation of HaloalkeneDerived Mercapturates. The mercapturates S-(1,1,2,2tetrafluoroethyl)-N-acetyl-L-cysteine, S-(2-chloro-1,1,2trifluoroethyl)-N-acetyl-L-cysteine, and S-(2-bromo-1,1,2trifluoroethyl)-N-acetyl-L-cysteine were deacetylated by acylase I. The acylase I specific activity with these substrates followed the order: S-(1,1,2,2-tetrafluoroethyl)-N-acetyl-L-cysteine > S-(2-chloro-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine > S-(2-bromo-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine. The kinetic data and calculated molar volumes and log P values are presented in Table 2. Examination of the relationship of acylase I specific activity with calculated molar volumes (Figure 4b) and log P (Figure 5b) values showed that the hydrolysis of the haloalkene-derived mercapturates decreased with increasing calculated molar volumes or log P values.

The Km and Vmax for the acylase I-catalyzed deacetylation of S-(2-chloro-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine and S-(2-bromo-1,1,2-trifluoroethyl)-N-acetyl-Lcysteine could not be determined because of substrate inhibition at substrate concentrations > 4 mM.

Discussion These results demonstrate that the deacetylation of NAC is catalyzed by acylase I. The enzyme catalyzing the deacetylation of NAC that was purified from rat kidney was similar to acylase I from porcine kidney (39) and bovine liver (40): both acylase I and the enzyme isolated in the present study had a subunit Mr of approximately 43 000, and both enzymes catalyzed the hydrolysis of NAC and NAM. The Km and Vmax for the deacetylation of NAC were 4.4 mM and 44.7 µmol/mg of protein/min, respectively. With NAM as the substrate, a Km of 2 mM has been reported for acylase I (39). n-Butylmalonic acid is a transition-state analogue of acylase I substrates and is a slow-binding inhibitor of


the porcine kidney acylase I-catalyzed deacetylation of NAM (41). n-Butylmalonic acid was also a slow-binding inhibitor of the acylase I-catalyzed deacetylation of NAC. Although the catalytic activities ascribed to acylase I are well-known, the physiological role of acylase I is poorly understood. It has been proposed that acylase I may participate in the salvage of N-acetylated amino acids (40, 42). Acylase I also catalyzes the hydrolysis of some N-acyl dipeptides (43). The activity of acylase I with a range of N-acyl-L-amino acids has been studied, and it has been shown that the electronegativity of the acyl group has a prominent effect on acylase I-catalyzed deacetylation reactions (44, 45). The present studies show that the length and degree of branching of the N-acyl-L-amino acid side chain also have prominent effects on acylase I-catalyzed deacetylation reactions. 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-Lcysteines with long (>C3) and branched S-alkyl substituents were poor acylase I substrates. The effect of chain length and degree of branching of the S-alkyl substituent on acylase I activity were related to calculated molar volumes. The data show that for substrates with unbranched S-alkyl substituents with chain lengths of C0C3, acylase I activity increased with increasing calculated molar volumes and then decreased with increasing calculated molar volumes for substrates with chain lengths >C3. The S-alkyl-N-acetyl-L-cysteines with branched S-alkyl substituents were poor acylase I substrates irrespective of their calculated molar volumes. The relationship between acylase I activity and calculated molar volumes indicates that the substrate binding site may be a relatively short and narrow pocket that cannot accommodate branched S-alkyl substituents and larger calculated molar volumes (chain length > C3). A similar relationship between acylase I activity and calculated molar volumes was not observed with N-acetylL-2-aminopentanoic acid, O-methyl-N-acetyl-DL-serine, and N-acetyl-L-2-aminohexanoic acid, which were poor acylase I substrates even though their calculated molar volumes were lower than those of the corresponding sulfur analogues. The observation that N-acetyl-L-2aminohexanoic acid is a competitive inhibitor of the acylase I-catalyzed deacetylation of NAM indicates that it binds to the enzyme but is not deacetylated by acylase I. N-Acetylseleno-L-methionine, the selenium analogue of NAM, has a slightly higher calculated molar volume than NAM and was a fairly good acylase I substrate. Thus, the presence of sulfur or selenium in the side chain of the N-acetyl-L-amino acid facilitates acylase I-catalyzed deacetylations. The acylase I specific activity was also related to calculated log P values. The relationship between acylase I specific activity and log P for NAC and the S-alkylN-acetyl-L-cysteines with unbranched S-alkyl substituents was similar to that observed with acylase I specific activity and calculated molar volumes. The S-alkyl-Nacetyl-L-cysteines with branched S-alkyl substituents, S-isopropyl-N-acetyl-L-cysteine, S-isobutyl-N-acetyl-Lcysteine, and S-tert-butyl-N-acetyl-L-cysteine, had lower log P values than their corresponding unbranched analogues but were poorer acylase I susbtrates. Furthermore, O-methyl-N-acetyl-DL-serine and N-acetyl-L-2aminopentanoic acid had lower and higher log P values, respectively, than the corresponding sulfur analogues but


were poorer acylase I susbstrates. Also, N-acetyl-L-2aminohexanoic acid had a higher log P value than its corresponding sulfur analogue but was a poorer acylase I substrate. It should be noted, however, that the log P values were calculated for un-ionized substrates, but ionization of the substrate is essential for binding (46, 47). The in vivo and in vitro deacetylation of mercapturates of nephrotoxic, cytotoxic, and mutagenic cysteine Sconjugates has been observed (19, 21), but the role of acylase I in these reactions has not been established. The acylase I-catalyzed deacetylation of mercapturates affords cysteine S-conjugates that may undergo β-lyasedependent bioactivation (20, 32). The present study shows that S-(1,1,2,2-tetrafluoroethyl)-N-acetyl-L-cysteine, S-(2-chloro-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine, and S-(2-bromo-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine were substrates for acylase I. Indeed, the specific activity of the acylase I-catalyzed deacetylation of these haloalkene-derived N-acetyl-L-cysteine S-conjugates was higher than that of halogen-lacking S-alkyl-N-acetyl-L-cysteines. The calculated molar volume of S-(1,1,2,2-tetrafluoroethyl)-N-acetyl-L-cysteine was similar to that of S-ethylN-acetyl-L-cysteine and N-acetyl-L-methionine, but its rate of deacetylation was about 5-fold higher. Also, the calculated molar volume of S-(2-bromo-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine was similar to that of S-isobutyl- and S-tert-butyl-N-acetyl-L-cysteine, but its rate of deacetylation was about 30-fold higher than that of S-isobutyl-N-acetyl-L-cysteine and S-tert-butyl-N-acetylL-cysteine did not undergo deacetylation. Furthermore, the calculated log P values of the haloalkene-derived N-acetyl-L-cysteine S-conjugates were considerably higher than the log P values of the S-alkyl-N-acetyl-L-cysteines, although within the series their rates of deacetylation decreased with increasing log P values. The relationship between acylase I specific activity and log P values for the haloalkene-derived N-acetyl-L-cysteines indicates that hydrophobicity may be important for substrate binding, but the results obtained in the present study need to be extended to establish a relationship between calculated molar volumes or log P values and rates of deacetylation. These results also indicate that acylase I plays an important role in the β-lyase-dependent bioactivation of haloalkenes and, therefore, contributes to the conjugation-dependent toxicity of haloalkenes. Although these results indicate a relationship between acylase I activity and calculated molar volumes, more data are needed determine whether calculated molar volumes may be used to predict acylase I activity with N-acyl-L-amino acids. Also, the finding that acylase I catalyzes the deacetylation of NAC combined with the knowledge that the electronegativity of the acyl group has a prominent effect on acylase I-catalyzed deacetylation reactions may be exploited for the design of more effective L-cysteine prodrugs. Immunohistochemical studies show that acylase I is localized in the proximal tubule of the kidney (48). This selective localization of acylase I may be used to target L-cysteine prodrugs to that region of the kidney. Thus, additional structure-activity studies are warranted that may lead to the design of N-acylL-cysteines that may be targeted for delivery of L-cysteine to specific tissues at effective rates. Finally, the finding that acylase I catalyzes the deacetylation of N-acetylseleno-L-methionine indicates that the N-acetyl derivatives of the Se-substituted-selenocysteine


conjugates may also be deacetylated by acylase I and that structure-activity relationships similar to those observed with the S-substituted-N-acetyl-L-cysteines may be observed. Se-Substituted-selenocysteine conjugates have been proposed as potential prodrugs to target antitumor agents to the kidney (49), and the cytotoxicity of several Se-substituted-selenocysteine conjugates has been studied (50). Further studies are warranted to study the targeting and efficacy of Se-substituted-N-acetylselenocysteine conjugates as potential antitumor agents.

Acknowledgment. The authors thank Ms. Sandra Morgan for her assistance in preparing the manuscript. This research was supported by National Institute of Environmental Health Sciences Grant ES03127.

References (1) Lauterburg, B. H., Corcoran, G. B., and Mitchell, J. R. (1983) Mechanism of action of N-acetylcysteine in the protection against the hepatotoxicity of acetaminophen in rats in vivo. J. Clin. Invest. 71, 980-991. (2) De Flora, S., Cesarone, C. F., Balansky, R. M., Albini, A., D’Agostini, F., Bennicelli, C., Bagnasco, M., Camoirano, A., Scatolini, L., Rovida, A., and Izzotti, A. (1995) Chemopreventive properties and mechanisms of N-acetylcysteine. The experimental background. J. Cell. Biochem. 58, 33-41. (3) Webb, W. R. (1962) Clinical evaluation of a new mucolytic agent acetylcysteine. J. Thorac. Cardiovasc. Surg. 44, 330-343. (4) Reas, H. W. (1963) The effect of N-acetylcysteine on the viscosity of tracheobronchial secretions in cystic fibrosis of the pancreas. J. Pediatr. 62, 31-35. (5) Aruoma, O. I., Halliwell, B., Hoey, B. M., and Butler, J. (1989) The antioxidant action of N-acetylcysteine: Its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid. Free Radical Biol. Med. 6, 593. (6) Dro¨ge, W., Eck, H.-P., and Mihm, S. (1992) HIV-Induced cysteine deficiency and T-cell dysfunction-A rationale for treatment with N-acetylcysteine. Immunol. Today 13, 211-214. (7) Witschi, A., Junker, E., Schranz, C., Speck, R. F., and Lauterburg, B. H. (1995) Supplementation of N-acetylcysteine fails to increase glutathione in lymphocytes and plasma of patients with AIDS. AIDS Res. Hum. Retroviruses 11, 141-143. (8) Thor, H., Molde´us, P., and Orrenius, S. (1979) Metabolic activation and hepatotoxicity. Effect of cysteine, N-acetylcysteine, and methionine on glutathione biosynthesis and bromobenzene toxicity in isolated rat hepatocytes. Arch. Biochem. Biophys. 192, 405413. (9) Sjo¨din, K., Nilsson, E., Hallberg, A., and Tunek, A. (1989) Metabolism of N-acetyl-L-cysteine. Some structural requirements for the deacetylation and consequences for the oral bioavailability. Biochem. Pharmacol. 38, 3981-3985. (10) Sheffner, A. L., Medler, E. M., Bailey, K. R., Gallo, D. G., Mueller, A. J., and Sarett, H. P. (1966) Metabolic studies with acetylcysteine. Biochem. Pharmacol. 15, 1523-1535. (11) Anders, M. W., and Dekant, W. (1994) Aminoacylases. Adv. Pharmacol. 27, 433-450. (12) Greenstein, J. P., and Winitz, M. (1961) Chemistry of the Amino Acids, Vol. 2, pp 1753-1767, John Wiley, New York. (13) Ko¨rdel, W., and Schneider, F. (1977) Renal aminoacylase, a zinc enzyme. Z. Naturforsch. 32c, 342-344. (14) Cook, R. M., Burke, B. J., Buchhagen, D. L., Minna, J. D., and Miller, Y. E. (1993) Human aminoacylase-I. Cloning, sequence, and expression analysis of a chromosome 3p21 gene inactivated in small cell lung cancer. J. Biol. Chem. 268, 17010-17017. (15) Mitta, M., Ohnogi, H., Yamamoto, A., Kato, I., Sakiyama, F., and Tsunasawa, S. (1992) The primary structure of porcine aminoacylase 1 deduced from cDNA sequence. J. Biochem. 112, 737742. (16) Jakob, M., Miller, Y. E., and Ro¨hm, K.-H. (1992) Cloning and sequence analyses of cDNAs encoding aminoacylase I from porcine kidney. Biol. Chem. Hoppe-Seyler 373, 1227-1231. (17) Suzuki, S., and Tateishi, M. (1981) Purification and characterization of a rat liver enzyme catalyzing N-deacetylation of mercapturic acid conjugates. Drug Metab. Dispos. 9, 573-577.

Uttamsingh et al. (18) Bray, H. G., and James, S. P. (1960) The formation of mercapturic acids. 4. Deacetylation of mercapturic acids by the rabbit, rat and guinea pig. Biochem. J. 74, 394-397. (19) Pratt, I. S., and Lock, E. A. (1988) Deacetylation and further metabolism of the mercapturic acid of hexachloro-1,3-butadiene by rat kidney cytosol in vitro. Arch. Toxicol. 62, 341-345. (20) Boogaard, P. J., Commandeur, J. N. M., Mulder, G. J., Vermeulen, N. P. E., and Nagelkerke, J. F. (1989) Toxicity of the cysteine-Sconjugates and mercapturic acids of four structurally related difluoroethylenes in isolated proximal tubular cells from rat kidney. Uptake of the conjugates and activation to toxic metabolites. Biochem. Pharmacol. 38, 3731-3741. (21) Commandeur, J. N. M., Stijntjes, G. J., Wijngaard, J., and Vermeulen, N. P. E. (1991) Metabolism of L-cysteine S-conjugates and N-(trideuterioacetyl)-L-cysteine S-conjugates of four fluoroethylenes in the rat. Role of balance of deacetylation and acetylation in relation to the nephrotoxicity of mercapturic acids. Biochem. Pharmacol. 42, 31-38. (22) Grenby, T. H., and L., Y. (1960) Biochemical studies of toxic agents. 12. The biosynthesis of n-propylmercapturic acid from n-propyl halides. Biochem. J. 75, 28-33. (23) Barnsley, E. A. (1964) The metabolism of S-methyl-L-cysteine in the rat. Biochim. Biophys. Acta 90, 24-36. (24) Thomson, A. E. R., Barnsley, E. A., and Young, L. (1963) Biochemical studies of toxic agents. 14. The biosynthesis of ethylmercapturic acid. Biochem. J. 86, 145-152. (25) Barnsley, E. A., Grenby, T. H., and Young, L. (1966) Biochemical studies of toxic agents. the metabolism of 1- and 2-bromopropane in rats. Biochem. J. 100, 282-288. (26) Bray, H. G., Franklin, T. J., and James, S. P. (1959) The formation of mercapturic acids. 3. N-Acetylation of S-substituted cysteines in the rabbit, rat and guinea pig. Biochem. J. 73, 465-473. (27) Pastuszak, J. J., and Chimiak, A. (1981) tert-Butyl group as thiol protection in peptide synthesis. J. Org. Chem. 46, 1868-1873. (28) Dymicky, M. (1976) N-Acetyl-L-tyrosine ethyl ester. Org. Prep. Proc. Int. 8, 219-222. (29) Kisfaludy, L., Mohacsi, T., Low, M., and Drexler, F. (1979) Pentafluorophenyl acetate: A new, highly selective acetylating agent. J. Org. Chem. 44, 654-656. (30) Marshall, R., Birnbaum, S. M., and Greenstein, J. P. (1956) Cobalt ion activation of renal acylase I. J. Am. Chem. Soc. 78, 46364642. (31) Synge, R. L. M. (1931) Experiments on amino acids. IV. The methyl ethers of some N-acetyl-hydroxyamino acids. Biochem. J. 33, 1931-1934. (32) Commandeur, J. N. M., Brakenhoff, J. P. G., De Kanter, F. J. J., and Vermeulen, N. P. E. (1988) Nephrotoxicity of mercapturic acids of three structurally related 2,2-difluoroethylenes in the rat. Biochem. Pharmacol. 37, 4495-4504. (33) Finkelstein, M. B., Vamvakas, S., Bittner, D., and Anders, M. W. (1994) Structure-mutagenicity and structure-cytotoxicity studies on bromine-containing cysteine S-conjugates and related compounds. Chem. Res. Toxicol. 7, 157-163. (34) Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. (35) Udenfriend, S., Stein, S., Bo¨hlen, P., Dairman, W., Leimgruber, W., and Weigele, M. (1972) Fluorescamine: A reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range. Science 178, 871-872. (36) Endo, Y. (1978) N-Acyl-L-aromatic amino acid deacylase in animal tissues. Biochim. Biophys. Acta 523, 207-214. (37) Morrison, J. F. (1982) The slow-binding and slow, tight-binding inhibition of enzyme-catalysed reactions. Trends Biochem. Sci. 7, 102-105. (38) Hansch, C., and Leo, A. (1979) Substituent Constants for Correlation Analysis in Chemistry and Biology, Wiley, New York. (39) Bruns, F. H., and Schulze, C. (1962) Acylase I. Reindarstellung, physikalisch-chemische Eigenschaften and Identifizieren mit “Hippurikase”. (Acylase I. Purification, physical-chemical properties and identification with hippurkase.) Biochem. Z. 336, 162181. (40) Gade, W., and Brown, J. L. (1981) Purification, characterization and possible function of R-N-acylamino acid hydrolase from bovine liver. Biochim. Biophys. Acta 662, 86-93. (41) Ro¨hm, K.-H. (1989) Butylmalonate is a transition state analogue for aminoacylase I. FEBS Lett. 250, 191-194. (42) Endo, Y. (1980) In vivo deacetylation of N-acetyl amino acids by kidney acylases in mice and rats. A possible role of acylase system in mammalian kidneys. Biochim. Biophys. Acta 628, 13-18. (43) Moravcsik, E., Telegdi, J., Tu¨do¨s, H., Ko¨mives, K., and O ¨ tvo¨s, L. (1977) Substrate specificity of acylase-I-catalyzed dipeptide hydrolysis. Acta Biochim. Biophys. Acad. Sci. Hung. 12, 399-402.

Acylase I-Catalyzed Hydrolysis of N-Acetyl-L-cysteines (44) Fones, W. S., and Lee, M. (1953) Hydrolysis of N-acyl derivatives of alanine and phenylalanine by acylase I and carboxypeptidase. J. Biol. Chem. 201, 847-856. (45) Chenault, H. K., Dahmer, J., and Whitesides, G. M. (1989) Kinetic resolution of unnatural and rarely occurring amino acids: Enantioselective hydrolysis of N-acyl amino acids catalyzed by acylase I. J. Am. Chem. Soc. 111, 6354-6364. (46) Henseling, J., and Ro¨hm, K.-H. (1988) Aminoacylase I from hog kidney: Anion effects and the pH dependence of kinetic parameters. Biochim. Biophys. Acta 959, 370-377. (47) Fu, S.-C. J., and Birnbaum. (1953) The hydrolytic action of acylase I on N-acylamino acids. J. Am. Chem. Soc. 75, 918-920. (48) Lo¨ffler, H. G., Schneider, F., Aumu¨ller, G., and Unsicker, K. (1982) Immunzytochemische Untersuchungen zur Lokalisation der Ami-

Chem. Res. Toxicol., Vol. 11, No. 7, 1998 809 noacylase I (EC 3.5.1.14) in der Schweineniere. (Immunocytochemical studies on the localization of aminoacylase I (EC 3.5.1.14) in pig kidney.) Acta Histochem. 25 (Suppl.), 57-60. (49) Andreadou, I., Menge, W. M. P. B., Commandeur, J. N. M., Worthington, E. A., and Vermeulen, N. P. E. (1996) Synthesis of novel Se-substituted selenocysteine derivatives as potential kidney selective prodrugs of biologically active selenol compounds: Evaluation of kinetics of β-elimination reaction in rat renal cytosol. J. Med. Chem. 39, 2040-2046. (50) Andreadou, I., van de Water, B., Commandeur, J. N. M., Nagelkerke, F. J., and Vermeulen, N. P. E. (1996) Comparative cytotoxicity of 14 novel selenocysteine Se-conjugates in rat renal proximal tubular cells. Toxicol. Appl. Pharmacol. 141, 287.

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