Acylase I-catalyzed deacetylation of N-acetyl-L-cysteine and S-alkyl-N- acetyl-L-cysteines

Article abstract of DOI:10.1021/tx980018b

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 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 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-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 poor 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.

Full text of DOI:10.1021/tx980018b

8
00
Chem. Res. Toxicol. 1998, 11, 800-809
Acyla se I-Ca ta lyzed Dea cetyla tion of N-Acetyl-L-cystein e
‡
a n d S-Alk yl-N-a cetyl-L-cystein es
†
§
,†
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 J anuary 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-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 M
identical with the M 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 of
92 ( 27 µM. These results show that acylase I catalyzes the deacetylation of NAC. The
r
of 43 000, which is
r
i
1
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 (C
substrates, whereas the S-alkyl-N-acetyl-L-cysteines with long (>C
0
-C
3
) and unbranched S-alkyl substituents were good acylase I
) and branched S-alkyl
3
substituents were poor 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.
In tr od u ction
NAC1 is an antioxidant with chemopreventive and
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
therapeutic effects and is used clinically for the manage-
ment 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). Al-
though 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 glu-
tathione synthesis in these cells (8). Also, human en-
dothelial cells, rat lung, intestinal, and liver homoge-
nates, and human liver homogenates catalyze the
deacetylation of NAC (9, 10).
ꢀ
(
EC 3.5.17, N -acetyl-L-lysine amidohydrolase), and acy-
lase 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 N -
acetyl-L-lysine, respectively. Acylase III shows a prefer-
ence for N-acyl aromatic amino acids (12).
Acylase I is well-characterized. Renal acylase I is a
zinc-containing, homodimeric protein with a subunit M
r
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-N-
acetyl-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 haloalkene-
derived, nephrotoxic, cytotoxic, and mutagenic cysteine
S-conjugates are deacetylated in vivo in rats and by rat
kidney cytosol and isolated renal proximal-tubular cells
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),
7
9].
*
Address correspondence to: M. W. Anders. Voice: 716-275-1681.
Fax: 716-244-9283. E-mail: anders@pharmacol.rochester.edu.
(19-21).
†
University of Rochester.
Haskell Laboratory.
Abbreviations: NAC, N-acetyl-L-cysteine; NAM, N-acetyl-L-me-
§
The present experiments were designed to identify and
characterize the aminoacylase that catalyzes the deacety-
lation of NAC and to study the effects of substrate
1
thionine; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel
electrophoresis.
S0893-228x(98)00018-6 CCC: $15.00 © 1998 American Chemical Society
Published on Web 06/19/1998

Acylase I-Catalyzed Hydrolysis of N-Acetyl-L-cysteines
Chem. Res. Toxicol., Vol. 11, No. 7, 1998 801
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 sub-
strates. Finally, the acylase-catalyzed deacetylation of
haloalkene-derived mercapturates of nephrotoxic, cyto-
toxic, and mutagenic cysteine S-conjugates was studied.
CH
.5 (m, 1H, (COOH)CH(NHCOCH
S-Isop r op yl-N-a cetyl-L-cystein e. 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
2
CH
3
), 2.0 (s, 1H, NHCOCH
3
), 2.8-3.1 (m, 2H, CHCH
3
2
S),
4
)).
by recrystallization from water. Pure white crystals were
1
obtained: mp 130 °C [lit. mp 134 °C (25)]; H NMR (D
2
O) δ 1.2
), 3.0 (m, 3H,
3
)).
(d of d, 6H, -CH(CH
3 2 3
) ), 2.0 (s, 3H, NHCOCH
CH -S-CH), 4.5 (m, 1, (COOH)CH(NHCOCH
2
S-n -Bu tyl-N-a cetyl-L-cystein e. 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
Ma ter ia ls a n d Meth od s
Ma ter ia ls. N-Acetyl-L-cysteine, L-cysteine, N-acetyl-L-me-
thionine, L-methionine, O-methyl-DL-serine, seleno-L-methion-
ine, 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
white crystals were obtained: mp 67-69 °C [lit. mp 67 °C (26)];
1
H NMR (D
2
O) δ 0.8 (t, 3H, SCH
2
CH
), 1.4-1.6 (m, 2H, SCH
CH ), 2.0 (s, 3H, NHCOCH
3
2
CH
2
CH
CH
), 2.8-3.1 (m, 2H,
)).
3
), 1.2-1.4 (m, 2H,
SCH
2H, SCH
CHCH
2
CH
2
CH
2
CH
3
2
2
CH CH ), 2.5 (t,
2
3
(
pentane-1,1-dicarboxylic acid), methyl iodide, ethyl iodide,
propyl iodide, isopropyl iodide, n-butyl iodide, isobutyl iodide,
-methyl-2-propanol, and 2-aminopentanoic acid (L-norvaline)
2
CH
2
CH
2
3
2
S), 4.5 (m, 1H, (COOH)CH(NHCOCH
3
2
S-Isobu tyl-N-a cetyl-L-cystein e. 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
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 ).
An a lytica l Meth od s. Melting points were determined with
recrystallization from water. Pure white crystals were ob-
1
1
a Mel-Temp melting point apparatus and are uncorrected.
H
tained: mp 134 °C; H NMR (D
2
O) for the new compound δ 0.9
CH(CH ), 2.0 (s, 3H,
CH(CH ), 2.8-3.1 (m, 2H,
), 4.5 (m, 1H, (COOH)CH(NHCOCH )). El-
emental Anal. Calcd for C S: C, 49.32; H, 7.76; N, 6.39.
Found: C, 49.46; H, 7.89; N, 6.57.
NMR spectra were recorded with a Bruker 270 MHz spectrom-
(d, 6H, CH(CH
NHCOCH ), 2.4 (d, 2H, CH
CHCH SCH (CH
3
)
2
), 1.4-1.6 (m, CH
2
3 2
)
1
eter operating at 270-MHz for H. Chemical shifts, δ, are
3
2
SCH
2
3 2
)
reported in parts per million (ppm). The HOD resonance at 4.7
2
2
3
)
2
3
1
ppm was used as the internal standard for H NMR spectra
9 3
H17NO
when D
ppm was used as the internal standard for H NMR spectra
when dimethyl sulfoxide-d was the solvent. Elemental analy-
2
O was the solvent. The solvent resonance peak at 2.47
1
S-ter t-Bu tyl-N-a cetyl-L-cystein e. S-tert-Butyl-N-acetyl-L-
cysteine was obtained by the acetylation of S-tert-butyl-L-
cysteine 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 dis-
solved 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 methanol-
6
ses were determined by Midwest Microlab (Indianapolis, IN).
Syn th eses. S-Alk yl-N-a cetyl-L-cystein es. S-Methyl-, S-
ethyl-, S-n-propyl-, S-isopropyl-, S-n-butyl-, and S-isobutyl-N-
acetyl-L-cysteines were synthesized as described by Grenby and
Young (22). The compounds were purified as described below.
S-Meth yl-N-a cetyl-L-cystein e. 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
1
water. Pure white crystals were obtained: mp 130 °C dec; H
NMR (DMSO-d
.0 (s, 3H, NHCOCH
(COOH)CH(NHCOCH
tal Anal. Calcd for C
6
) for the new compound δ 1.4 (s, 9H, C(CH
), 2.8-3.0 (m, 2H, CHCH S), 4.4 (m, 1H,
)), 8.2-8.4 (d, 1H, NHCOCH ). Elemen-
S: C, 49.32; H, 7.76; N, 6.39.
3 3
) ),
2
3
2
1
3
3
(
23)]; H NMR (D
CH SCH ), 2.8-3.1 (m, 2H, CHCH
COOH)CH(NHCOCH )).
S-Eth yl-N-a cetyl-L-cystein e. The reaction mixture was
2
O) δ 2.0 (s, 3H, NHCOCH
3
), 2.1 (s, 3H,
SCH ), 4.5-4.6 (m, 1H,
3
9
H17NO
3
2
3
2
Found: C, 49.75; H, 8.00; N, 6.72.
(
3
N-Acetyl-L-2-a m in op en ta n oic Acid , N-Acetyl-O-m eth yl-
DL-ser in e, a n d N-Acetylselen o-L-m eth ion in e. These were
obtained by the acetylation of L-2-aminopentanoic acid, O-
methyl-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.
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
1
°
C (24)]; H NMR (D
2
O) δ 1.2 (t, 3H, CH
), 2.5 (q, 2H, CH SCH CH ), 2.8-3.1 (m, 2H, CHCH
), 4.5-4.6 (m, 1H, (COOH)CH(NHCOCH )).
2 2 3
SCH CH ), 2.0 (s, 3H,
NHCOCH
SCH CH
3
2
2
3
2
-
2
3
3
N-Acetyl-L-2-a m in op en ta n oic Acid . The product gave
1
white crystals: mp 97-99 °C [lit. mp 100 °C (30)]; H NMR
S-P r op yl-N-a cetyl-L-cystein e. 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
(
D
2
O) δ 0.75 (t, 3H, CHCH
CHCH CH CH ), 1.6-1.8 (m, 2H, CHCH
NHCOCH ), 4.2 (m, 1H, (COOH)CH(NHCOCH
2 2 3
CH CH ), 1.25-1.4 (m, 2H,
2
2
3
2
CH
2
CH
3
), 2.0 (s, 3H,
3
3
)).
O-Meth yl-N-a cetyl-DL-ser in e. The product gave a yellow-
ish-white powder: mp 105-107 °C [lit. mp 108-109 °C (31)];
1
product was recrystallized from water. Pure white crystals were
H NMR (D
2
O) δ 2.0 (s, 3H, NHCOCH
3 2 3
), 3.3 (s, 3H, CH OCH ),
1
obtained: mp 96 °C [lit. mp 96 °C (22)]; H NMR (D
2
O) δ 0.9 (t,
3.65-3.9 (m, 2H, CH
COCH )).
2
OCH ), 4.5 (m, 1H, (COOH)CH(NH-
3
3
H, SCH
2
CH
2
CH
3
), 1.5 (m, 2H, SCH
2
CH
2
CH
3
), 2.5 (t, 2H, SCH
2
-
3

8
02 Chem. Res. Toxicol., Vol. 11, No. 7, 1998
Uttamsingh et al.
N-Acetylselen o-L-m eth ion in e. The product was obtained
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 spectro-
photometer (Norwalk, CT). A standard curve was prepared with
L-methionine.
1
as a white powder: mp 107 °C; H NMR (D
2
O) for the new
), 2.45-
Se), 4.4-4.5 (m, 1H, (COOH)CH(NH-
)). Elemental Anal. Calcd for C Se: C, 35.30;
compound δ 1.9 (s, 3H, SeCH
.7 (m, 4H, CHCH CH
COCH
3 3
), 2.0 (s, 3H, NHCOCH
2
2
2
3
7 3
H13NO
H, 5.46; N, 5.88. Found: C, 35.41; H, 5.58; N, 5.83.
S-(1,1,2,2-Tetr aflu or oeth yl)-N-acetyl-L-cystein e an d S-(2-
Br om o-1,1,2-tr iflu or oeth yl)-N-acetyl-L-cystein e. These were
prepared by the acetylation of S-(1,1,2,2-tetrafluoroethyl)-L-
cysteine (32) and S-(2-bromo-1,1,2-trifluoroethyl)-L-cysteine (33),
respectively, with pentafluorophenyl acetate in DMF, as de-
scribed 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 recrys-
tallized from ethyl acetate-hexane. S-(1,1,2,2-Tetrafluoroet-
hyl)-N-acetyl-L-cysteine was obtained as a white powder; its
physical constants were identical with the reported values (32).
L-Cysteine formed by the deacetylation of NAC was deter-
mined 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).
S-(2-Bromo-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine was ob-
1
tained as a white powder: mp 103-104 °C; H NMR (D
2
O) for
the new compound δ 2.0 (s, 3H, NHCOCH
CHCH SCF ), 4.5-4.6 (m, 1H, (COOH)CH(NHCOCH
.8, 6.9-7.0 (d of t, 1H, CH SCHF CHBrF). Elemental Anal.
Calcd for C NO SBrF : C, 26.01; H, 2.48; N, 4.33. Found:
C, 26.40; H, 2.88; N, 4.26.
3
), 3.2-3.5 (m, 2H,
2
2
3
)), 6.7-
6
2
2
7
H
8
3
3
S-(2-Ch lor o-1,1,2-tr iflu or oeth yl)-N-acetyl-L-cystein e. This
was prepared as described by Boogaard et al. (20).
En zym e P u r ifica tion . Kidneys from male Sprague-Daw-
ley 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
Acylase I activity with NAC, NAM, S-methyl-, S-ethyl-, S-n-
propyl-, 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, N-acetylseleno-L-methionine, O-methyl-
N-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-(2-
bromo-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-L-
cysteines, N-acetylseleno-L-methionine, S-(1,1,2,2-tetrafluoro-
ethyl)-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 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-acetyl-
DL-serine were incubated for 1 h at 37 °C. The amounts of
L-amino acids formed were measured as described above.
1
0 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 Ion-
D, 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. Frac-
tions 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 con-
centrated in an Amicon ultrafiltration cell, and 5 mL of the
concentrated fraction was applied to a Phenyl-Sepharose column
Kin etic An a lyses. 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
m
the K 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.
[
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 (NH SO . Proteins
4
)
2
4
m
Experiments were performed in triplicate. The K and Vmax
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 stain-
ing. 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.
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
3
7 °C, and 50-µL samples were withdrawn at 3-min intervals
Activity Assa ys. Acylase I activity was determined by
measuring the formation of deacetylated L-amino acids. L-
Methionine formed by the deacetylation of NAM was determined
by reaction with fluorescamine (35). The reaction mixture (1
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. K
i
values were determined from the progress curves as described

Acylase I-Catalyzed Hydrolysis of N-Acetyl-L-cysteines
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 ) V
s
t + (V
)/k. Kinetic constants were computed by the EZ-FIT pro-
gram.
The inhibition of the acylase I-catalyzed deacetylation of NAM
o
- V )(1 -
s
-
kt
e
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.
Ca lcu la tion s. 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
3
cm × 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.
Resu lts
En zym e P u r ifica tion . Rat kidney cytosol was frac-
tionated by ammonium sulfate precipitation, DEAE-
cellulose column chromatography, Sephacryl S-200 col-
umn 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 col-
umn. The acylase in fractions 22 and 23 from the Phenyl-
Sepharose column showed a purification of 200- and 289-
fold with NAC and NAM, respectively, as the substrates
(
Figure 1C) (Table 1). Fractions from the Phenyl-
F igu r e 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
Sepharose column were analyzed by SDS-PAGE, which
showed that the protein was purified to near homogeneity
and had a M
purified protein and commercial acylase I had identical
values when analyzed by SDS-PAGE.
The K and Vmax for the rat kidney acylase I-catalyzed
r
of 43 000 (Figure 2). In addition, the
(
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. Frac-
tions 134-206 had high aminoacylase activity. (B) Sephacryl
S-200 HR column chromatography of fractions 134-206 ob-
tained from DEAE-cellulose column chromatography. The col-
umn was eluted with 10 mM potassium phosphate buffer (pH
M
r
m
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 K 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
k
because of large coefficients of variation.
Str u ctu r e-Activity Stu d ies. The acylase I-cata-
lyzed deacetylation of NAC, a range of S-alkyl-N-acetyl-
L-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 vol-
umes 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
i
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 aminoacy-
lase 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
off and kon values could not be satisfactorily determined
4 2 4
phosphate buffer (pH 7.0) containing 1.7 M (NH ) SO (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 activi-
ties 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 sub-
strates followed the order: NAC < S-methyl-N-acetyl-L-

8
04 Chem. Res. Toxicol., Vol. 11, No. 7, 1998
Ta ble 1. P u r ifica tion of Am in oa cyla se fr om Ra t Kid n ey
Uttamsingh et al.
N-Acetyl-L-cysteine
N-Acetyl-L-methionine
protein
concn
(mL) (mg/mL)
total
protein
(mg)
specific
activity
(nmol/mg/min) (nmol/min)
total
activity
specific
activity
(nmol/mg/min) (nmol/min)
total
activity
vol
purification
(fold)
purification
(fold)
fraction
cytosol
-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
60
180
343
1377
12000
580500
95040
64827
68850
2640
45
160
337
1117
13000
435375
84480
63693
55850
2860
0
3.0
5.7
23
3.6
7.5
25.0
289.0
0.22
200
oxygen analogues of S-methyl-N-acetyl-L-cysteine, was
lower than with S-methyl-N-acetyl-L-cysteine. No deacety-
lation 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 K
i
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 deacety-
lation 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 S-
alkyl substituents increased with increasing molar vol-
ume (NAC < S-methyl-N-acetyl-L-cysteine < S-ethyl-N-
acetyl-L-cysteine) and then decreased with further
increases in molar volumes (S-n-propyl-N-acetyl-L-cys-
teine > S-n-butyl-N-acetyl-L-cysteine). For the S-alkyl-
N-acetyl-L-cysteines with branched-chain S-alkyl sub-
stituents, 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 straight-
chain analogues. Thus, S-n-propyl-N-acetyl-L-cysteine
and S-isopropyl-N-acetyl-L-cysteine had identical calcu-
lated molar volumes, but acylase I activity with S-
isopropyl-N-acetyl-L-cysteine was lower than activity
with S-n-propyl-N-acetyl-L-cysteine. Similarly, S-n-but-
yl-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-N-
acetyl-L-cysteine was lower than with S-n-butyl-N-acetyl-
L-cysteine; activity with S-tert-butyl-N-acetyl-L-cysteine
was not detected. Acylase I activity with O-methyl-N-
acetyl-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.
F igu r e 2. Electrophoretic analysis of purified rat kidney
acylase I. Fractions from the Phenyl-Sepharose column chro-
matography step (see Figure 1C) were analyzed by SDS-PAGE,
as described in Materials and methods. Fractions were 3-8 and
1
9-23. Fractions 22 and 23 and commercial porcine kidney
acylase I (lane AI) had identical M
r
.
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). S-
Isopropyl-N-acetyl-L-cysteine, S-isobutyl-N-acetyl-L-cys-
teine, 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-acetyl-
L-2-aminopentanoic acid was lower than with the sulfur-
containing 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.
F igu r e 3. Inhibition of the acylase I-catalyzed deacetylation
of NAC by n-butylmalonate. The reaction mixtures contained
5
0
3 units of pig kidney acylase I, 2 mM NAC, and 0 (b), 0.2 (9),
.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-N-
acetyl-L-cysteine
>
S-n-butyl-N-acetyl-L-cysteine
>
S-isopropyl-N-acetyl-L-cysteine ≈ S-isobutyl-N-acetyl-L-
cysteine. No deacetylation of S-tert-butyl-N-acetyl-L-
cysteine 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

Acylase I-Catalyzed Hydrolysis of N-Acetyl-L-cysteines
Chem. Res. Toxicol., Vol. 11, No. 7, 1998 805
Ta ble 2. Rela tion sh ip betw een Mola r Volu m e or log P a n d Kin etics of S-Su bstitu ted -N-a cetyl-L-cytsein es
specific activitya
molar
volume
(µmol/mg of
Km
Vmax
Vmax/Km
substrate
log P
-0.642
protein/min)
(mM) (µmol/mg/min) (mL/mg/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
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
19.9 ( 3.2
4.4
2.1
>10.0
3.4
44.7
70.9
NDc
95.2
ND
87.1
45.5
ND
18.5
ND
ND
ND
58.4
ND
10.2
33.8
-0.301
0.436
0.228
0.965
-0.487
0.757
0.537
1.286
1.066
0.936
-1.063
27.1 ( 4.7
3.1 ( 0.5
33.1 ( 3.1
28.0
activity not detected >10.0
30.8 ( 3.0
24.7 ( 1.2
1.8 ( 1.3
5.1 ( 1.5
1.5 ( 1.1
2.1
0.8
>10.0
3.7
41.5
56.9
5.0
>10.0
activity not detected >10.0
4.7 ( 0.5b
>10.0
4.0
ND
ND
ND
N-acetylseleno-L-methionine
not available 14.9 ( 1.9
14.6
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
1.560
1.900
2.040
161 ( 11.3
68 ( 2.3
51.6 ( 5.2
ND
ND
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.
F igu r e 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-(1,1,2,2-
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. (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-tetrafluo-
roethyl)-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 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-methyl-N-acetyl-DL-serine were
incubated for 1 h. Enzyme activity was measured as described in Materials and Methods.

8
06 Chem. Res. Toxicol., Vol. 11, No. 7, 1998
Uttamsingh et al.
F igu r e 5. (a) Relationship between specific activity and calculated log P values of NAC, NAM, S-alkyl-N-acetyl-L-cysteines, O-methyl-
N-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-chloro-
1
,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-methyl-
N-acetyl-DL-serine were incubated for 1 h. Enzyme activity was measured as described in Materials and Methods.
The relationship between Vmax/ K
molar volumes and between Vmax/ K and log P values is
shown in Table 2. S-n-Propyl-N-acetyl-L-cysteine exhib-
ited the highest Vmax/ K
m
and calculated
m
The K and Vmax for the acylase I-catalyzed deacety-
m
lation of S-(2-chloro-1,1,2-trifluoroethyl)-N-acetyl-L-cys-
teine and S-(2-bromo-1,1,2-trifluoroethyl)-N-acetyl-L-
cysteine could not be determined because of substrate
inhibition at substrate concentrations > 4 mM.
m
.
Acyla se I-Ca ta lyzed Dea cetyla tion of Ha loa lk en e-
Der ived Mer captu r ates. The mercapturates 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 deacetylated by
acylase I. The acylase I specific activity with these
substrates followed the order: S-(1,1,2,2-tetrafluoro-
ethyl)-N-acetyl-L-cysteine > S-(2-chloro-1,1,2-trifluoro-
ethyl)-N-acetyl-L-cysteine > S-(2-bromo-1,1,2-trifluoro-
ethyl)-N-acetyl-L-cysteine. The kinetic data and calcu-
lated molar volumes and log P values are presented in
Table 2.
Discu ssion
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 M
approximately 43 000, and both enzymes catalyzed the
hydrolysis of NAC and NAM. The K and Vmax for the
r
of
m
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.
deacetylation of NAC were 4.4 mM and 44.7 µmol/mg of
protein/min, respectively. With NAM as the substrate,
a K
m
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

Acylase I-Catalyzed Hydrolysis of N-Acetyl-L-cysteines
Chem. Res. Toxicol., Vol. 11, No. 7, 1998 807
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).
were poorer acylase I susbstrates. Also, N-acetyl-L-2-
aminohexanoic 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 S-
conjugates 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 af-
fords cysteine S-conjugates that may undergo â-lyase-
dependent bioactivation (20, 32). The present study
shows that S-(1,1,2,2-tetrafluoroethyl)-N-acetyl-L-cys-
teine, 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 halo-
alkene-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-tetrafluoroet-
hyl)-N-acetyl-L-cysteine was similar to that of S-ethyl-
N-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-trifluoro-
ethyl)-N-acetyl-L-cysteine was similar to that of S-isobut-
yl- 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-acetyl-
L-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 â-lyase-dependent bioac-
tivation 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 deacetyla-
tion reactions may be exploited for the design of more
effective L-cysteine prodrugs. Immunohistochemical stud-
ies 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 stud-
ies are warranted that may lead to the design of N-acyl-
L-cysteines that may be targeted for delivery of L-cysteine
to specific tissues at effective rates.
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 deacety-
lation reactions. S-Alkyl-N-acetyl-L-cysteines with short
(
C
0
-C
acylase I substrates, whereas the S-alkyl-N-acetyl-L-
cysteines with long (>C ) and branched S-alkyl substit-
3
) and unbranched S-alkyl substituents were good
3
uents 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 un-
branched S-alkyl substituents with chain lengths of C
0
-
C
3
, acylase I activity increased with increasing calculated
molar volumes and then decreased with increasing
calculated molar volumes for substrates with chain
lengths >C
3
.
The S-alkyl-N-acetyl-L-cysteines with
branched S-alkyl substituents were poor acylase I sub-
strates irrespective of their calculated molar volumes.
The relationship between acylase I activity and calcu-
lated molar volumes indicates that the substrate binding
site may be a relatively short and narrow pocket that
cannot accommodate branched S-alkyl substituents and
3
larger calculated molar volumes (chain length > C ). A
similar relationship between acylase I activity and
calculated molar volumes was not observed with N-acetyl-
L-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-2-
aminohexanoic 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 acy-
lase I specific activity and log P for NAC and the S-alkyl-
N-acetyl-L-cysteines with unbranched S-alkyl substitu-
ents was similar to that observed with acylase I specific
activity and calculated molar volumes. The S-alkyl-N-
acetyl-L-cysteines with branched S-alkyl substituents,
S-isopropyl-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 ana-
logues but were poorer acylase I susbtrates. Further-
more, O-methyl-N-acetyl-DL-serine and N-acetyl-L-2-
aminopentanoic acid had lower and higher log P values,
respectively, than the corresponding sulfur analogues but
Finally, the finding that acylase I catalyzes the deacety-
lation of N-acetylseleno-L-methionine indicates that the
N-acetyl derivatives of the Se-substituted-selenocysteine

8
08 Chem. Res. Toxicol., Vol. 11, No. 7, 1998
Uttamsingh et al.
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 ob-
served. 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 stud-
ied (50). Further studies are warranted to study the
targeting and efficacy of Se-substituted-N-acetylseleno-
cysteine conjugates as potential antitumor agents.
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Ack n ow led gm en t. 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.
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Immunzytochemische Untersuchungen zur Lokalisation der Ami-
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