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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics
JPET 294:753–761, 2000 /2467/838126
Vol. 294, No. 2
Printed in U.S.A.
Bioactivation of Selenocysteine Se-Conjugates by a Highly
Purified Rat Renal Cysteine Conjugate ␤-Lyase/Glutamine
Transaminase K1
JAN N. M. COMMANDEUR, IOANNA ANDREADOU,2 MARTIJN ROOSEBOOM, MARCEL OUT, LAURENS J. DE LEUR,
ED GROOT, and NICO P. E. VERMEULEN
Leiden/Amsterdam Center for Drug Research, Division of Molecular Toxicology, Department of Pharmacochemistry, Vrije Universiteit
Amsterdam, Amsterdam, the Netherlands
Accepted for publication April 19, 2000
This paper is available online at http://www.jpet.org
The conjugation of electrophilic substrates to glutathione
(GSH) and subsequent disposition of the GSH S-conjugates
formed are mediated by a large number of different enzymes
and transport systems present in various tissues (Commandeur et al., 1995). One of the pathways involved in the catabolism of GSH-conjugates is the ␤-elimination reaction of
the corresponding cysteine S-conjugates by cysteine conjugate ␤-lyases (␤-lyases), resulting in the formation of ammonia, pyruvic acid, and thiol compounds. In the case of glutathione S-conjugates of halogenated alkenes, the thiols formed
by ␤-lyase may rearrange rapidly to chemically highly reactive intermediates, such as thionoacyl halides, thiiranes,
and/or thioketenes (Dekant et al., 1988; Commandeur et al.,
Received for publication January 6, 2000.
1
Financial support was provided by the European Science Foundation.
2
I.A. was a visiting scientist from School of Pharmacy of the Aristotelian
University of Thessaloniki, Greece.
cated by the rapid consumption of ␣-keto-␥-methiolbutyric
acid, purified ␤-lyase/GTK also catalyzed transamination reactions, which appeared to even exceed that of ␤-elimination. The
corresponding sulfur analogs also showed significant transamination but were ␤-eliminated at an extremely low rate. Comparison of the obtained enzyme kinetic data of purified ␤-lyase/
GTK with previously obtained data from rat renal cytosol
showed a poor correlation. By determining the activity profiles
of cytosolic fractions applied to anion exchange fast protein
liquid chromatography and gel filtration chromatography, the
involvement of multiple enzymes in the ␤-elimination of selenocysteine Se-conjugates in rat renal cytosol was demonstrated.
The identity and characteristics of these alternative selenocysteine conjugate ␤-lyases, however, remain to be established.
1996). The combination of active uptake mechanisms and a
relatively high activity of ␤-lyase in the kidney may explain
the relatively selective nephrotoxicity of many halogenated
alkenes in rodents. A number of studies by the group of
Elfarra have demonstrated that the biochemical basis of this
kidney selectivity may also be applied to target pharmacologically active thiol-containing antitumor agents, such as
mercaptopurine and thioguanine, to the kidney for the treatment of renal cell carcinoma (Hwang and Elfarra, 1989, 1991;
Elfarra and Hwang, 1993; Elfarra et al., 1995). More recently, the structurally strongly related selenocysteine Seconjugates were proposed as alternative prodrugs to target
pharmacologically selenol compounds to the kidney by local
␤-lyase enzyme systems (Andreadou et al., 1996).
Although the above-mentioned prodrugs appear to be activated by renal subcellular fractions, the enzymes that are
actually involved in these ␤-elimination reactions have not
ABBREVIATIONS: GSH, glutathione; ␤-lyase, cysteine S-conjugate ␤-lyase; GTK, glutamine transaminase K; KMB, ␣-keto-␥-methiolbutyric acid;
OPD, o-phenylene diamine; FPLC, fast protein liquid chromatography; BTC, S-(benzothiazolyl)-L-cysteine; 1,2-DCV-Cys, S-(1,2-dichlorovinyl)-Lcysteine; TFE-Cys, S-(1,1,2,2-tetrafluoroethyl)-L-cysteine; PMSF, phenylmethylsulfonyl fluoride; CTFE-Cys, S-(2-chloro-1,1,2-trifluoroethyl)-Lcysteine; DCDFE-Cys, S-(2,2-dichloro-1,1-difluoroethyl)-L-cysteine; MALDI-TOF, matrix-assisted laser desorption time-of-flight.
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ABSTRACT
Selenocysteine Se-conjugates have recently been proposed as
potential prodrugs to target pharmacologically active selenol
compounds to the kidney. Although rat renal cytosol displayed
a high activity of ␤-elimination activity toward these substrates,
the enzymes involved in this activation pathway as yet have not
been identified. In the present study, the possible involvement
of cysteine conjugate ␤-lyase/glutamine transaminase K (␤lyase/GTK) in cytosolic activity was investigated. To this end,
the enzyme kinetics of 15 differentially substituted selenocysteine Se-conjugates and 11 cysteine S-conjugates was determined using highly purified rat renal ␤-lyase/GTK. The results
demonstrate that most selenocysteine Se-conjugates are
␤-eliminated at a very high activity by purified ␤-lyase/GTK,
implicating an important role of this protein in the previously
reported ␤-elimination reactions in rat renal cytosol. As indi-
754
Commandeur et al.
Experimental Procedures
Materials. ␣-Keto-␥-methiolbutyric acid (KMB), ␤-chloro-L-alanine, S-methyl-L-cysteine, S-ethyl-L-cysteine, S-benzyl-L-cysteine,
and a diagnostic kit for aspartate aminotransferase (DG158-K) were
purchased from Sigma Chemical Co. (St. Louis, MO). Amino-oxyacetic acid and phenylmethylsulfonyl fluoride (PMSF) were obtained
from Aldrich Chemie (Brussels, Belgium). 4-Methoxythiophenol was
obtained from Fluka (Buchs, Switzerland). S-(4-Methylbenzyl)-L-cysteine was purchased from Advanced Chemtech (Louisville, KY). S-(4Methoxybenzyl)-L-cysteine was purchased from Bachem Feinchemikalien AG (Bubendorf, Switzerland).
3
Due to a typing error, Abraham and Cooper (1996) originally reported a
mass of Mr 45,800 for this protein. The correct mass of the protein, based on its
cDNA sequence, is Mr 48,500 (D. G. Abraham, personnel communication).
Fig. 1. Concept of activation of cysteine S-conjugates and selenocysteine
Se-conjugates to pharmacologically active thiols and selenols, respectively.
Selenocysteine Se-conjugates and L-selenocystine were prepared
as described by Andreadou et al. (1996). S-Allyl-L-cysteine was prepared according to Freeman et al. (1994). S-(1,2-Dichlorovinyl)-Lcysteine (1,2-DCV-Cys) was synthesized as described by McKinney
et al. (1959). S-(1,1,2,2-Tetrafluoroethyl)-L-cysteine (TFE-Cys), S-(2chloro-1,1,2-trifluoroethyl)-L-cysteine (CTFE-Cys), and S-(2,2-dichloro-1,1-difluoroethyl)-L-cysteine (DCDFE-Cys) were synthesized
as described by Commandeur et al. (1988).
Synthesis of Se-Allyl-L-selenocysteine. L-Selenocystine (1.5
mmol, 500 mg) was dissolved in 8 ml of 0.5 N NaOH and 2 ml of
ethanol. At 0°C, 0.4 g (15 mmol) of NaBH4 was added while the
reaction mixture was stirred. The mixture was allowed to reach room
temperature, during which the color of the solution changed from
yellow to colorless, After cooling again to 0°C, 4 ml of 2 N NaOH and
6 mmol of allylbromide were added, and the mixture was stirred for
3 h at room temperature. Concentrated HCl was added until pH 5 to
6 and cooled at 4°C. Se-Allyl-L-cysteine precipitated as a yellowish
solid. The 1H NMR spectrum obtained was almost identical with that
of S-allyl-L-cysteine (17): 1H NMR (D2O, Na2CO3): ␦ (ppm) 2.95 to
3.22 (2H, m, CH2-CH-NH2), 3.30 (2H, d, CH2ACH-CH2), 4.25 to 4.40
(1H, d of d, CH2-CH-NH2), 5.05 to 5.25 (2H, m, CH2ACH-CH2), 5.80
to 6.08 (1H, m, CH2ACH-CH2).
Purification of Rat Renal Cysteine Conjugate ␤-Lyase/GTK.
␤-Lyase/GTK was purified from rat renal cytosol obtained from male
Wistar rats (160 –250 g) supplied by Harlan (Zeist, the Netherlands).
The procedure used is described in detail by Yamauchi et al. (1993)
and yielded a highly purified enzyme that was 1000-fold enriched
according to its increased specific activity with S-(1,2-dichlorovinyl)L-cysteine (1,2-DCV-Cys) as a substrate. Matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry analysis of
this protein revealed a single MH⫹ mass of Mr 47,700 ⫾ 130,4 which
is consistent with the mass as predicted from the sequence of ␤-lyase/
GTK as determined by Perry et al. (1993).
The purified ␤-lyase/GTK obtained was dissolved in 50 mM TrisHCl buffer, pH 8.0, and was stored in 100-␮l fractions of 100 ␮g/ml
at ⫺20°C. When stored under these conditions, no significant decrease in specific activity toward 1,2-DCV-Cys was observed after 2
years.
Enzymatic Incubations. Selenocysteine Se-conjugates and cysteine S-conjugates were incubated with the purified ␤-lyase in 50
mM Tris-HCl buffer, pH 8.6, and at a temperature of 37°C. Unless
stated otherwise, the final concentration of enzyme was 1 ␮g/ml. The
cofactor KMB was added at a final concentration of 0.2 mM, which
allows assessment of transamination reaction by measuring KMB
consumption (see later). After 2 min of preincubation at 37°C, incubations were started by the addition of a 10-fold concentrated enzyme solution. ␤-Elimination reactions were assessed by determining the formation of pyruvic acid. To correct for background
production of pyruvic acid, parallel incubations were always performed in absence of enzyme. All incubations were always performed
in duplicate.
Specific activities of transamination of all conjugates were determined at a substrate concentration of 0.5 mM. Transamination re4
MALDI-TOF mass spectrometry measurements were carried out on a
VISION 2000 (Finnigan MAT, Bremen, Germany) (for details, see Jespersen et
al., 1995). Samples containing 125 fmol of ␤-lyase/GTK in 2,5-dihydroxybenzoic acid as matrix were irradiated with a nitrogen laser at 337 nm. Mass
spectra were accumulated for 25 laser shots fired at the same spot on the
sample surface.
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yet been identified. To elucidate the possibilities and limitations of this prodrug concept, however, the identity and tissue distribution of the enzymes involved in the activation of
cysteine S-conjugates and selenocysteine Se-conjugates remain to be characterized.
Three major cysteine conjugate ␤-lyase enzymes have been
identified in rat kidney cytosol (Cooper, 1998). All are pyridoxal 5⬘-phosphate-dependent enzymes. One cytosolic
␤-lyase in the kidney appeared to be identical with glutamine
transaminase K (GTK), based on its composition and enzyme
kinetic properties (Stevens et al., 1986). Originally denoted
as GTK, this enzyme was isolated more than 25 years ago
(Cooper and Meister, 1974). ␤-Lyase/GTK is a dimeric protein consisting of two identical subunits of Mr 47,470. Recent
cloning studies revealed that ␤-lyase/GTK is also identical
with kynurenine aminotransferase (Perry et al., 1993; Mosca
et al., 1994). More recently, a closely related cysteine conjugate ␤-lyase, with subunits of a slightly higher3 molecular
weight, 48,500, was characterized with considerable overlap
in substrate selectivity (Abraham and Cooper, 1996). Finally,
a ␤-lyase enzyme with high molecular weight (330,000) was
demonstrated to be present in both cytosolic and mitochondrial fractions. This third ␤-lyase has different enzyme characteristics compared with ␤-lyase/GTK as demonstrated by
its ability to convert leukotriene E4 and 5⬘-S-cysteinyldopamine and by its lower specific activity toward cysteine conjugates of halogenated alkenes (Abraham et al., 1995).
Recently, we demonstrated that replacing the sulfur of
cysteine S-conjugates by a selenium atom resulted in a dramatic increase in ␤-elimination activity in rat renal cytosol
(Andreadou et al., 1996). Therefore, selenocysteine Se-conjugates were proposed as alternative prodrugs to target pharmacologically active selenol compounds to the kidney (Fig. 1).
The aim of the present investigation was to study whether
selenocysteine Se-conjugates are substrates for purified rat
renal ␤-lyase/GTK. Next to the selenocysteine Se-conjugates,
a number of corresponding cysteine S-conjugates were tested
as substrates for purified rat renal ␤-lyase/GTK as well.
Because the enzyme kinetics obtained with the purified enzyme showed a relatively poor correlation with results previously obtained with rat renal cytosol, the possible involvement of multiple enzymes was studied by determining the
activity profiles after fractionation of rat renal cytosol by two
different chromatographic methods: anion exchange chromatography and gel permeation chromatography.
Vol. 294
2000
755
cytosolic protein) was applied to the Mono-Q column, which was
equilibrated with the same buffer as used for dialysis.
The column was eluted at a flow rate of 1 ml/min. After an initial
10 min of elution with buffer A, a linear gradient was started by
mixing with buffer B (buffer A containing 900 mM sodium chloride).
Fifty minutes after the start of the gradient, the eluent reached 100%
buffer B. During chromatography, elution of proteins was monitored
by UV detection at 280 nm. From the start of the FPLC, 40 fractions
of 2 ml were collected and stored at ⫺20°C until analysis for enzyme
activity.
Activity profiles of ␤-elimination were determined by incubating
all fractions with five different substrates: CTFE-Cys, Se-phenyl-Lselenocysteine, Se-allyl-L-selenocysteine, Se-isopropyl-L-selenocysteine, and Se-(4-methylbenzyl)-L-selenocysteine. Then, 15 ␮l of the
FPLC fractions was added to 135 ␮l of 50 mM Tris-HCl buffer, pH
8.6, containing 2 mM substrate and 0.2 mM KMB. Incubations were
performed for 20 min at 37°C, after which the reaction was terminated by adding 500 ␮l of 12 mM OPD in 3 N HCl. After 60 min of
derivatization at 60°C, the samples were analyzed by HPLC, as
described earlier.
Elution of aspartate aminotransferase, which was previously proposed as an alternative ␤-eliminating enzyme (Kato et al., 1996), was
monitored spectrophotometrically by coupling oxaloacetate formation to NADH oxidation with malate dehydrogenase, using the diagnostic kit from Sigma Chemical Co.
Fractionation of Rat Kidney Cytosol by Gel Permeation
Chromatography. Rat renal cytosol (5 ml) was applied to a HiLoad
16/60 Superdex 200 column (Pharmacia Biotech) and eluted at a flow
rate of 0.5 ml/min with 50 mM sodium phosphate buffer, pH 7.4,
containing 150 mM sodium chloride and 40 ␮M PMSF. After 65 min,
representing the time necessary to elute the dead volume, fractions
of 0.5 ml were collected and placed on ice. Elution of proteins was
monitored continuously by UV detection at 280 nm. Activities were
determined using Se-(4-methylbenzyl)-L-selenocysteine and CTFECys as substrates. Then, 15 ␮l of the fractions was added to 135 ␮l of
50 mM Tris-HCl buffer, pH 8.6, containing substrate and 0.5 mM
KMB. Incubations were performed for 20 min at 37°C, after which
the reaction was terminated by adding 500 ␮l of 12 mM OPD in 3 M
HCl. After 60 min of derivatization at 60°C, the samples were analyzed by HPLC, as described earlier.
Retention times of gel permeation column were calibrated by
eluting a mixture of marker proteins of known molecular weight.
Observed retention times were ␣-lactalbumin (14,200 Da), 302 min;
carbonic anhydrase (29,000 Da), 257 min; chicken egg albumin
(45,000 Da), 188 min; BSA monomer (66,000 Da), 177 min; BSA
dimer (132,000 Da), 161 min; urease trimer (272,000 Da), 146 min;
and urease hexamer (545,000 Da), 125 min.
Results
Time Course of Biotransformation of Cysteine
S-Conjugates and Selenocysteine Conjugates. A significant time-dependent formation of pyruvic acid was observed
on incubation of purified ␤-lyase/GTK with cysteine S-conjugates of halogenated alkenes and with various selenocysteine
Se-conjugates (Fig. 2). Because pyruvate formation significantly deviated from linearity after approximately 5 min, the
enzyme kinetic parameters Km and kcat for the conjugates
were based on the specific activities obtained using incubation times of 5 min.
In the present incubation systems, the cofactor KMB is
added to reactivate ␤-lyase/GTK, which is converted to the
PMP form by a concurrent transamination-reaction route
(Stevens et al., 1986). The rate of KMB consumption therefore reflects the rate of transamination reactions. As shown
in Fig. 1, the cofactor KMB is consumed during the incuba-
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actions was assessed by determining consumption of the ␣-keto acid
cofactor KMB as described previously (Cooper and Meister, 1974;
Stevens et al., 1986). This approach precludes the necessity to develop standards and analytical assays for all individual ␣-keto acid
products formed from the conjugates studied. Both pyruvic acid and
KMB can be assessed in a single assay after derivatization with the
␣-keto acid-reagent o-phenylene diamine (OPD), followed by analysis
by HPLC equipped with fluorescence detection (Stijntjes et al.,
1992). In the case of selenocysteine Se-conjugates, specific activities
of the transaminase pathway were determined by incubation for 5
min at 37°C in presence of 1 ␮g/ml ␤-lyase/GTK (final concentration).
To obtain significant KMB consumption with cysteine S-conjugates,
these compounds were incubated for 30 min at 37°C in presence of 2
␮g/ml ␤-lyase/GTK.
Time Course of Enzyme Reactions. Before determining enzyme kinetics, the time course of product formation was determined
to assess the linearity of the ␤-elimination reactions. To this end,
incubations were performed at an incubation volume of 2 ml. After
starting the reaction by the addition of 200 ␮l of 10 ␮g/ml enzyme
solution, samples of 100 ␮l were taken from the incubation at several
time points and mixed with 500 ␮l of OPD solution (12 mM OPD in
3 M HCl) in 1-ml Eppendorf cups. The Eppendorf cups were closed
and heated for 60 min at 60°C to complete the derivatization reaction
(Stijntjes et al., 1992). To the resulting solutions, 600 ␮l of HPLC
eluent (45% methanol, 54% water, 1% acetic acid) was added and
transferred to HPLC vials for automated HPLC analysis (see later).
Enzyme Kinetic Parameters. Enzyme kinetic parameters (Km
and kcat) of conjugates were determined by incubating substrates at
six to eight concentrations ranging from 0.05 to 4 mM. Due to their
poor solubility, the conjugates with phenyl and benzyl substituents
were incubated at concentrations up to 2 mM. Corrections for nonenzymatic degradation were made by performing parallel incubations in absence of enzyme. All incubations were performed at 37°C
and at a total volume of 300 ␮l and were started by adding 30 ␮l of
10 ␮g/ml purified ␤-lyase/GTK. The final concentration of KMB in all
incubations was 0.2 mM. Reactions were stopped after 5 min of
incubation by the addition of 1 ml of 12 mM OPD in 3 M HCl. The
resulting mixtures were subsequently heated for 60 min at 60°C.
After this derivatization reaction, a volume of 600 ␮l was mixed with
600 ␮l of HPLC eluent (45% methanol, 54% water, 1% acetic acid)
and transferred to HPLC vials for automated HPLC analysis (see
later).
HPLC Analysis of Pyruvic Acid and KMB. HPLC vials were
placed in a Waters 707 Autoinjector cooled at 4°C. Then, 100-␮l
samples were injected at intervals of 20 min. The analytes were
chromatographed on two ChromSpher C18 columns (5-␮m particles,
100 ⫻ 4.6 mm; Chrompack, Bergen op Zoom, The Netherlands),
which was eluted isocratically with the above-mentioned HPLC
eluent at a flow rate of 0.4 ml/min. Detection of derivatized pyruvic
acid and KMB was accomplished with a Jasco fluorescence detector
(model 821-FP) set at an excitation wavelength of 336 nm and an
emission wavelength of 420 nm. Chromatograms were analyzed using the Class VP 4.1 software package of Shimadzu (Columbia, MD).
Under these conditions, retention times of derivatized pyruvic acid
and KMB were 5.8 and 14.9 min, respectively.
For quantification of the analytes, calibration curves were constructed by derivatizing known concentrations (ranging from 5 to
200 ␮M) of pyruvic acid and KMB in 50 mM Tris-HCl, pH 8.6, with
12 mM OPD in 3 M HCl. After heating for 60 min at 60°C, samples
were treated and analyzed in the same manner as described above.
Fractionation of Rat Kidney Cytosol by Anion Exchange
Fast Protein Liquid Chromatography (FPLC). Rat kidney cytosol was fractionated by FPLC using a Mono-Q anion exchange
column (Pharmacia Biotech, Uppsala, Sweden). Before application to
the column, rat kidney cytosol was dialysed for 20 h against 20 mM
triethanol-HCl buffer, pH 7.45, containing 0.1 mM EDTA and 40 ␮M
PMSF (buffer A). After dialysis, this enzyme fraction was filtered
using a 0.2-␮m Schlauer filter, and 0.5 ml (containing 19 mg of
Bioactivation of Se-Conjugates by ␤-Lyase/GTK
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Commandeur et al.
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Fig. 2. Time course of pyruvic acid formation and KMB consumption in incubation of purified cysteine conjugate ␤-lyase/GTK (1 ␮g/ml) with 1 mM
cysteine S-conjugates (top) or 1 mM selenocysteine Se-conjugates (middle and bottom). F, pyruvic acid; E, KMB. Error bars represent difference
between two data points.
tions to a different extent. In the incubations with TFE-Cys
and CTFE-Cys as substrates, the concentration of KMB was
only decreased from 200 ␮M (initial concentration) to 175 to
180 ␮M. In the incubations of the selenocysteine Se-conjugates, however, the consumption of KMB appeared to be
significantly higher, leading to a decrease of more than 70%
during a 30-min incubation period.
Substrate Selectivity and Kinetics of Purified
␤-Lyase/GTK. The specific activities and enzyme kinetic
parameters of purified ␤-lyase/GTK toward 11 cysteine S-
Bioactivation of Se-Conjugates by ␤-Lyase/GTK
2000
757
TABLE 1
Enzyme kinetic parameters of purified rat renal cysteine conjugate beta-lyase/glutamine transaminase K toward differentially substituted
cysteine S-conjugates (X ⫽ S) and selenocysteine Se-conjugates (X ⫽ Se)
Beta-Elimination Reaction
X-Bound Substituent
Specific Activity
Methyl
Ethyl
n-Propyl
i-Propyl
n-Butyl
Allyl
TFE
CTFE
DCDFE
1,2-DCV
Benzyl
4-Methoxybenzyl
4-Chlorobenzyl
3,4-Dichlorobenzyl
Phenyl
4-Methylphenyl
4-Methoxyphenyl
4-Chlorophenyl
Se
S
Se
S
Se
Se
Se
Se
S
S
S
S
S
Se
S
Se
S
Se
S
Se
Se
Se
S
Se
Se
Se
a
Km
kcat
kcat/Km
Specific Activitya
nmol/min 䡠 mg
mM
min⫺1
min⫺1 䡠 mM⫺1
nmol/min 䡠 mg
1610 ⫾ 40
N.D.
3580 ⫾ 210
122 ⫾ 35
4330 ⫾ 380
4410 ⫾ 400
1430 ⫾ 520
4790 ⫾ 630
146 ⫾ 34
11,240 ⫾ 3120
6260 ⫾ 860
4230 ⫾ 290
4120 ⫾ 460
1110 ⫾ 570
20 ⫾ 12
10,220 ⫾ 3210
N.D.
8300 ⫾ 1210
42 ⫾ 22
3780 ⫾ 1800
9190 ⫾ 1500
9130 ⫾ 1300
53 ⫾ 30
136 ⫾ 12
25 ⫾ 15
65 ⫾ 10
5.0 ⫾ 2.5
N.D.
3.0 ⫾ 1.2
N.D.
0.94 ⫾ 0.31
0.44 ⫾ 0.16
0.26 ⫾ 0.05
1.19 ⫾ 0.23
N.D.
1.24 ⫾ 0.31
0.72 ⫾ 0.25
0.51 ⫾ 0.24
0.51 ⫾ 0.19
0.81 ⫾ 0.34
N.D.
0.66 ⫾ 0.21
N.D.
0.66 ⫾ 0.21
N.D.
0.70 ⫾ 0.04
0.64 ⫾ 0.18
0.92 ⫾ 0.20
N.D.
N.D.
N.D.
N.D.
1790 ⫾ 480
N.D.
2500 ⫾ 950
N.D.
990 ⫾ 110
790 ⫾ 90
210 ⫾ 150
1950 ⫾ 230
N.D.
3520 ⫾ 520
1460 ⫾ 160
985 ⫾ 120
987 ⫾ 89
249 ⫾ 32
N.D.
2270 ⫾ 340
N.D.
2270 ⫾ 340
N.D.
1170 ⫾ 450
2020 ⫾ 640
2430 ⫾ 360
N.D.
N.D.
N.D.
N.D.
340 ⫾ 30
N.D.
850 ⫾ 100
N.D.
1200 ⫾ 360
1810 ⫾ 230
810 ⫾ 290
1640 ⫾ 230
N.D.
3090 ⫾ 210
2030 ⫾ 145
1669 ⫾ 162
1930 ⫾ 250
406 ⫾ 150
N.D.
3430 ⫾ 450
N.D.
3170 ⫾ 190
N.D.
1670 ⫾ 320
3160 ⫾ 120
2740 ⫾ 490
N.D.
N.D.
N.D.
N.D.
7400 ⫾ 1550
1370 ⫾ 250
11,100 ⫾ 1500
1200 ⫾ 175
13,100 ⫾ 1500
24,500 ⫾ 1750
9500 ⫾ 2000
18,000 ⫾ 1900
1500 ⫾ 300
5060 ⫾ 1000
6600 ⫾ 1500
8500 ⫾ 1400
9500 ⫾ 2100
3200 ⫾ 1200
850 ⫾ 180
18,200 ⫾ 2200
2100 ⫾ 230
18,000 ⫾ 2500
1330 ⫾ 250
7100 ⫾ 2000
13,000 ⫾ 3500
21,000 ⫾ 3100
750 ⫾ 150
320 ⫾ 100
420 ⫾ 120
600 ⫾ 200
N.D., not detectable.
a
Specific activity at 0.5 mM substrate concentration.
conjugates and 15 selenocysteine Se-conjugates as substrates
are shown in Table 1. From these results, it appears that
most cysteine S-conjugates tested showed only a very low
␤-elimination activity. Significant consumption of KMB was
observed, indicative for a preference for the transamination
reaction. The only L-cysteine S-conjugates showing a sufficiently high ␤-elimination activity enabling assessment of
enzyme kinetic parameters were the four cysteine S-conjugates carrying halogenated alkyl and alkenyl substituents. A
decrease in specific activity and kcat/Km was observed in the
order TFE-Cys ⬎ CTFE-Cys ⬎ DCDFE-Cys ⬇ 1,2-DCV-Cys.
These nephrotoxic cysteine S-conjugates also appear to be
transaminated to a significant extent as indicated by a significant consumption of KMB (Table 1). The relative importance of transamination appears to slightly increase in the
order TFE-Cys ⬍ CTFE-Cys ⬍ DCDFE-Cys ⬇ 1,2-DCV-Cys.
Compared with their sulfur analogs, the selenocysteine
Se-conjugates appeared to be metabolized at much higher
activities by purified cysteine conjugate ␤-lyase/GTK (Table
1). The ␤-elimination activities of several selenocysteine Seconjugates were almost equivalent to that of TFE-Cys, the
best ␤-elimination substrate for this enzyme known as yet.
Furthermore, transamination appears to be an important
pathway of metabolism of selenocysteine Se-conjugates, because KMB consumption was always significantly higher
than pyruvic acid production. Of three selenocysteine Seconjugates, the D-selenocysteine Se-conjugates were also
tested: Se-(methyl)-D-selenocysteine, Se-(n-propyl)-D-selenocysteine, and Se-(4-methylbenzyl)-D-selenocysteine. For
these stereoisomers, however, no significant ␤-elimination or
KMB consumption was observed, indicative of absolute stereoselectivity of ␤-lyase/GTK for the L-isomers. The pyruvic
acid formation and KMB consumption of all substrates
shown in Table 1 could be blocked completely by 1 mM
amino-oxyacetic acid.
The specific activities of ␤-elimination of selenocysteine
conjugates with n-alkyl-substituents were 30 to 50 times
higher than that of the corresponding cysteine S-conjugates,
enabling Lineweaver-Burke analyses. An increase in the
length of the n-alkyl-chain appears to increase the affinity for
␤-lyase/GTK, because the Michaelis-Menten constant Km decreases significantly from 5 mM (methyl substituent) to 0.26
mM (n-butyl-substituent). In contrast, kcat values decrease
with increased chain length. Se-(n-Butyl)-L-selenocysteine
showed substrate inhibition at concentrations higher than
0.5 mM. Therefore, the enzyme kinetic parameters for this
substrate were estimated from the activities obtained below
0.5 mM.
Se-Allyl-L-selenocysteine and S-allyl-L-cysteine, compounds known to occur in garlic (Lu et al., 1996), both showed
␤-elimination on incubation with purified ␤-lyase/GTK (Table 1). Again, the selenium compound showed a 30-fold
higher specific activity at a concentration of 0.5 mM than its
sulfur analog. For S-allyl-L-cysteine, enzyme activities were
too low to allow determination of enzyme kinetic parameters
Km and kcat. With Se-allyl-L-selenocysteine as substrate,
KMB consumption was almost 4-fold higher than pyruvic
acid formation, again indicative for extensive concurrent
transamination. KMB consumption was also observed in incubations with S-allyl-L-cysteine although at an approximately 10-fold lower rate compared with its selenium analog.
Selenocysteine Se-conjugates with benzyl substituents also
showed high ␤-elimination activities (Table 1). Interestingly,
para-substitution at the benzyl group strongly increased en-
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4-Methylbenzyl
Transamination Reaction
X
758
Commandeur et al.
the initial 0.2 mM KMB was consumed during incubation. As
was the case with Se-phenyl-L-selenocysteine and Se-(4methylbenzyl)-L-selenocysteine, pyruvic acid formation in
fraction 10 was almost 2-fold higher at a 0.5 mM KMB
concentration, resulting in an activity profile comparable
with that of CTFE-Cys.
To localize the protein ␤-lyase/GTK in the FPLC fractions,
Western blotting was performed using serum of rabbits immunized with the purified rat ␤-lyase/GTK. Rabbit antiserum raised against highly purified rat renal ␤-lyase/GTK was
kindly provided by Dr. A. Yamauchi (Kobe University, Japan).
A very strong cross-reactivity, with a mass identical with to of
the purified ␤-lyase/GTK, was observed in fraction 10. Of all
other fractions, only a weak staining was observed in fraction
11 (data not shown). Fractions 12 to 18, which showed significant ␤-elimination activity and KMB consumption, apparently
did not contain the ␤-lyase/GTK protein at all.
Activity of aspartate aminotransferase was present only in
fractions 8 and 9, with a 5-fold higher activity in fraction 8
(data not shown). The fact that fraction 8 does not show
␤-elimination activity indicates that aspartate aminotransferase is not involved in the ␤-elimination activity of the
conjugates studied.
Fractionation of Rat Kidney Cytosol by Gel Filtration Chromatography. Rat renal cytosol was also fractionated by high-resolution gel filtration chromatography using a
HiLoad 16/60 Superdex 200 column. By using Se-(4-methylbenzyl)-L-selenocysteine and CTFE-Cys as substrates, activity profiles were determined. As shown in Fig. 4, at least two
broad peaks with ␤-elimination activity were shown. For
both substrates, maximal activity was present in fraction 20,
which eluted 150 min after the application of renal cytosol to
the column. According to the calibration by the marker protein mixture, the mass of this protein will be around Mr
300,000. The second peak had its highest activity in fraction
37, which eluted after 167 min. According to the calibration
by the marker protein mixture, the mass of this protein will
between Mr 66,000 and 132,000, which may be consistent
with the 90-kDa ␤-lyase protein or proteins.
Discussion
Local activation of cysteine S-conjugates by renal ␤-eliminating enzymes has been proposed as a novel approach to
target antitumor compounds 6-mercaptopurine and 6-thioguanine to the kidney (Hwang and Elfarra, 1989, 1991). More
recently, selenocysteine Se-conjugates were proposed as alternative kidney-selective prodrugs showing much higher
␤-elimination activities in rat renal cytosol than their corresponding sulfur analogs (Andreadou et al., 1996). However,
as yet little is known regarding which proteins are actually
involved in the ␤-elimination reactions of these S- and Seconjugates because activities have been measured only in
crude enzyme fractions such as kidney homogenates, mitochondria, and cytosols.
One of the enzymes capable of catalyzing transamination
and ␤-elimination reactions is ␤-lyase/GTK. It has been suggested that large noncharged amino acids are transaminated
by ␤-lyase/GTK (Cooper and Meister, 1974). Using purified
␤-lyase/GTK from rat kidney, next to transamination, a high
␤-elimination activity has been demonstrated with 1,2-DCVCys and TFE-Cys as substrates. No ␤-elimination activity
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zyme activities compared with the unsubstituted Se-benzylL-selenocysteine; ␤-elimination activities almost equaled
those of TFE-Cys. Because KMB consumption even exceeds
pyruvic acid formation, these results suggest that overall
activity (i.e., ␤-elimination plus transamination) of the substituted Se-benzyl-L-selenocysteines is even higher than that
of TFE-Cys. No clear differences were observed between electron-withdrawing (chloro substituents) and electron-donating (methyl and methoxy substituents) substituents, suggesting that steric or lipophilic effects are more important
than electronical effects.
Se-phenyl-L-selenocysteine also is a very good substrate for
␤-lyase/GTK, as indicated by its high activities of pyruvic
acid production and KMB consumption. The specific activity
of Se-phenyl-L-selenocysteine at 0.5 mM is more than 150fold higher than that of S-phenyl-L-cysteine (Table 1), again
pointing to the superiority of selenocysteine Se-conjugates
over cysteine S-conjugates as substrates for ␤-lyase/GTK.
However, in contrast to the benzyl-substituted selenocysteine-Se-conjugates, the introduction of para-substituents at
the phenyl ring of Se-phenyl-L-selenocysteine results in a
dramatic decrease in enzyme activity (Table 1). To generate
sufficient pyruvic acid, incubation with these substrates were
performed for 20 min and in presence of a 10-fold higher
concentration of purified ␤-lyase/GTK. Se-(4-methylphenyl)L-selenocysteine and Se-(4-chlorophenyl)-L-selenocysteine
demonstrated substrate inhibition at concentrations higher
than 0.5 mM. Se-(4-methoxyphenyl)-L-selenocysteine was the
selenocysteine Se-conjugates displaying the lowest pyruvic
acid formation (Table 1).
Fractionation of Rat Kidney Cytosol by Anion Exchange Chromatography. Rat renal cytosol was fractionated using Mono-Q anion exchange FPLC, and the 2-ml fractions collected were subsequently screened for ␤-lyase
activity using five different substrates. Using CTFE-Cys as a
substrate, the highest activity was found in fraction 10,
whereas lower activities were found in fractions 11, 12, and
13 (Fig. 3A). In incubations of fractions with CTFE-Cys as a
substrate, only a relatively small consumption of KMB was
observed (⬍20% of the initial 0.2 mM concentration). When
using Se-phenyl-L-selenocysteine and Se-(4-methylbenzyl)-Lselenocysteine as substrate, however, a significantly different activity profile was observed, which rules out involvement of only a single enzyme in the ␤-elimination reactions.
First, in contrast to CTFE-Cys, maximal activity was observed in fraction 11 (Fig. 3, B and C). Second, using these
selenocysteine conjugates, very significant pyruvic acid formation was also observed in fractions 14 to 18. Furthermore,
with both substrates, a much stronger, up to 60%, consumption of KMB was observed, which again was significant from
fractions 10 to 18. To test whether KMB might have become
limiting in these experiments, the activity of these fractions
were also determined in presence of 0.5 mM KMB. Interestingly, at this higher KMB concentration, the highest pyruvate formation was observed in fraction 10, as is the case
with CTFE-Cys as substrate (data not shown).
Se-(isopropyl)-L-selenocysteine (Fig. 3D) and Se-allyl-L-selenocysteine (data not shown), at 0.2 mM KMB, had almost
identical activity profiles with maximal activity in fractions
10, 11, and 12 and low but still significant activities in
fractions 13 to 18. With these selenocysteine Se-conjugates,
KMB consumption was even more significant; up to 80% of
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2000
Bioactivation of Se-Conjugates by ␤-Lyase/GTK
759
was observed using S-(benzothiazolyl)-L-cysteine (BTC)
(Yamauchi et al., 1993), 5⬘-S-cysteinyldopamine and leukotriene E4 as substrates (Abraham et al., 1995). In the present
study, nine additional cysteine S-conjugates were evaluated
as substrates for purified renal ␤-lyase/GTK. As shown in
Table 1, high ␤-elimination activities were also observed with
CTFE-Cys and DCDFE-Cys as substrate. In accordance with
the observations by Stevens et al. (1986) using 1,2-DCV-Cys,
significant transaminase activity was also observed as implicated by the relatively high rates of KMB consumption that
were observed.
The present study confirms that cysteine S-conjugates carrying nonhalogenated substituents are poor substrates in
comparison with their selenium analogs (Table 1). Enzymatic
pyruvic acid production was only 25 to 50% of the low nonenzymatic degradation of the conjugates (data not shown).
Only at a higher enzyme concentration and with a longer
incubation time was significant consumption of KMB observed for these S-conjugates, indicative of a preference for
transamination reactions. Although the present nonhalogenated cysteine S-conjugates have not yet been tested with the
purified rat ␤-lyase/GTK, some of them have been tested
previously as substrates for purified ␤-lyases from bovine
and turkey kidney (Bhattacharya and Schultze, 1967). Consistent with the present study, no ␤-elimination was previously observed with S-methyl-L-cysteine, S-ethyl-L-cysteine,
S-propyl-L-cysteine, S-benzyl-L-cysteine, and S-allyl-L-cysteine. Lash et al. (1990) reported that BTC and S-(benzothiazolyl)-L-homocysteine were actively ␤-eliminated by a purified ␤-lyase from human kidney. However, Yamauchi et al.
(1993) did not find any activity of purified rat renal ␤-lyase/
GTK toward BTC, suggesting a significant species difference
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Fig. 3. Activity profiles of ␤-elimination reactions (indicated by pyruvate formation) and transamination reactions (as indicated by KMB consumption)
with CTFE-Cys and three selenocysteine Se-conjugates in FPLC fractions from rat renal cytosol applied to a Mono-Q anion exchange column. Activities
were expressed as percentage of the highest peak area of pyruvate and KMB, respectively.
760
Commandeur et al.
in substrate selectivity between the human and rat enzymes.
This species difference is further supported by the overall
18% dissimilarity between the amino acid sequences of the
rat and human enzyme (Perry et al., 1993).
Selenocysteine Se-conjugates previously were shown to be
␤-eliminated at a high activity by renal cytosol (Andreadou et
al., 1996). The results of the present study suggest that
␤-lyase/GTK plays a major role in this reaction, because most
Se-conjugates were ␤-eliminated at a very high activity. Several selenocysteine Se-conjugates displayed ␤-elimination activities as high as that observed with TFE-Cys, which was
the best substrate known. As was observed previously in rat
renal cytosol, the activity of ␤-elimination of Se-conjugates
was much higher than ␤-elimination of corresponding Sconjugates (Table 1). Possible explanations for the higher
␤-elimination reactions of selenocysteine Se-conjugates may
be the weaker bond strength of the C-Se-bond (234 kJ/mol)
versus C-S-bonds (272 kJ/mol) (Guziec, 1987) and/or a more
facilitated ␤-proton abstraction of the selenocysteine moiety
(Miles, 1986). As indicated by the significant KMB consumption, transamination reaction is a prominent pathway of
biotransformation of both selenocysteine Se-conjugates and
cysteine S-conjugates. When comparing specific activities of
transamination, selenocysteine Se-conjugates as substrates
showed 5- to 10-fold higher activities compared with their
sulfur analogs (Table 1). Because transamination reactions
do not involve C-Se-scission, facilitation of ␤-proton abstraction by electronic effects of the Se atom may be the most
likely explanation.
Because only very few substrates have been identified as
yet, little was known regarding the structure-activity relationship of ␤-lyase/GTK-catalyzed ␤-elimination reactions.
Because of their surprisingly high activities, the class of
selenocysteine Se-conjugates may be interesting probe substrates to characterize the substrate-binding site. For the
alkyl-substituted Se-conjugates, an increase in the alkylchain appears to increase the affinity for the enzyme, as
indicated by the decrease in their Km value (Table 1). The
benzyl-substituted Se-conjugates appeared to be extremely
good substrates as well, especially when substituted at the
para-position of the benzyl group. No clear electronic effects
were observed because both electron-withdrawing and electron-donating para-substituents appeared to increase
␤-elimination activity compared with Se-benzyl-L-selenocysteine. Therefore, apparently steric properties or lipophilicity
plays a more important role. Surprisingly, the introduction of
para-substituents in Se-phenyl-L-selenocysteine led to a very
strong decrease in enzyme activities (Table 1).
When comparing enzyme kinetic parameters of the purified ␤-lyase/GTK with those obtained previously with rat
renal cytosol, a very poor correlation is found. The most
striking difference is found with the para-substituted Sephenyl-L-selenocysteine compounds, which showed high activity with rat renal cytosol (Andreadou et al., 1996) but
demonstrated only a minor activity with purified ␤-lyase/
GTK. By determining the activity profile in fractionated rat
renal cytosol, the present study reveals that the poor correlation between cytosolic and purified enzyme activities may
be explained by the involvement of multiple enzymes in the
cytosolic fractions (Figs. 3 and 4). Aspartate aminotransferase, which was proposed by Kato et al. (1996) as an alternative rat renal ␤-lyase enzyme, does not appear to be involved
in ␤-elimination of the conjugates tested. According to the
results of the gel filtration chromatography, an important
role may be played by the high-molecular-weight ␤-lyase
previously characterized by Abraham et al. (1995).
Recently, it was shown that selenium-enriched garlic was
more effective in cancer prevention than normally grown
garlic (Lu et al., 1996). Among the selenium species identified
in selenium-enriched garlic were two of the selenocysteine
Se-conjugates, Se-methyl-L-selenocysteine and Se-allyl-L-selenocysteine, tested in this study. Dietary administration of
these selenocysteine Se-conjugates to rats indeed provided
strong protection against methylnitrosourea-induced carcinogenesis in rats (Ip et al., 1999). Chemopreventive activities
of these selenocysteine Se-conjugates have been attributed to
methyl selenol (Ip and Ganther, 1992) and diallyl selenide
(Lu et al., 1996), respectively, both of which are formed via
␤-elimination reactions. The relatively high activity of purified ␤-lyase/GTK toward these selenocysteine Se-conjugates
(Table 1) may implicate a role of ␤-lyase/GTK in the chemopreventive activity of selenium-enriched garlic. Diallyl selenide was reported to have a 100-fold higher chemopreventive activity than diallyl sulfide, which is an important
chemopreventive agent in normally grown garlic (el-Bayoumy et al., 1996). Therefore, the combination of higher bioactivation activity with formation of a 100-fold more potent
product may explain the higher chemopreventive activity of
Se-allyl-L-selenocysteine compared with S-allyl-L-cysteine.
In conclusion, the results presented here indicate that rat
renal ␤-lyase/GTK plays an important role in the ␤-elimination of selenocysteine Se-conjugates by rat renal cytosol. The
high ␤-elimination activity in combination with the potent
antitumor activities of the formed selenol compounds makes
selenocysteine Se-conjugates promising prodrugs to treat renal cell carcinoma. Fractionation of renal cytosol by two
different types of chromatography, however, revealed that
next to ␤-lyase/GTK, additional enzymes are active in the
␤-elimination of selenocysteine Se-conjugates. The identity
and tissue distribution of these additional enzymes remain to
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Fig. 4. Activity profiles of ␤-elimination reactions (indicated by pyruvate
formation) with CTFE-Cys (E) and Se-(4-methylbenzyl)-L-selenocysteine
(F) in FPLC fractions from rat renal cytosol applied to a HiLoad 16/60
Superdex 200 gel filtration column.
Vol. 294
2000
be established to predict the kidney selectivity of selenocysteine Se-conjugates as prodrugs.
Acknowledgments
Dr. S. Jespersen (TNO Nutrition and Food Research) at the Department of Bio-Pharmaceutical Analysis (Zeist, the Netherlands) is
gratefully acknowledged for performing the MALDI-TOF mass spectrometry measurements on ␤-lyase/GTK.
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Bioactivation of Se-Conjugates by ␤-Lyase/GTK