Chem. Res. Toxicol. 1988, I , 175-178
175
Thioacylating Intermediates as Metabolites of S-( 1,2-Dichlorovinyl)-~-cysteine and S-( 1,2,2-Trichlorovinyl)-~-cysteineFormed by Cysteine Conjugate @-Lyase Wolfgang Dekant,? Klemens Berthold,+S p y r i d o n Vamvakas,+Dietrich Henschler,'
and M. W. Anders**f
Institut f u r Toxikologie, Universitat Wiirzburg, Versbacher Str. 9, 0-8700 Wiirzburg, Federal Republic of Germany, and Department of Pharmacology, University of Rochester, 601 Elmwood Avenue, Rochester, New York 14642 Received February 17, 1988 T h e bioactivation mechanism of S-(1,2-dichlorovinyl)-~-cysteine (DCVC) and S-(1,2,2-trichloroviny1)-L-cysteine (TCVC) was studied with cysteine conjugate 0-lyase (P-lyase) from Salmonella t y p h i m u r i u m and with the pyridoxal phosphate model N-dodecylpyridoxal bromide (PL-Br) as catalysts and with G C / M S t o identify the metabolites formed. PL-Br converted S-2-benzothiazolyl-~-cysteine t o 2-mercaptobenzothiazole and S-benzyl-L-cysteine t o benzyl mercaptan, demonstrating the ability of PL-Br to serve as a model for @-lyase. PL-Br and bacterial 0-lyase converted DCVC to chloroacetic acid and chlorothionoacetic acid and TCVC to dichloroacetic acid. Incubations of PL-Br with the S-conjugates in the presence of diethylamine resulted in the formation of N,N-diethylchlorothioacetamidefrom DCVC and of N,N-diethyldichlorothioacetamide from TCVC. Attempts to trap the enethiols, which are the expected initial products formed by 0-elimination, by reaction with methyl iodide in incubations with the @-lyasemodel were not successful. T h e formation of thioacylating agents from the enethiols may contribute to the cytotoxic and mutagenic effects of DCVC and TCVC.
Introduction Cysteine S-conjugates are intermediates in the metabolism of glutathione S-conjugates to mercapturic acids (1-3). Several halogenated alkenes are conjugated with glutathione by hepatic glutathione S-transferases, and, for these compounds, glutathione conjugation initiates a sequence of events which results in nephrotoxicity (1-3). Cysteine conjugate 0-lyase (@-lyase')catalyzed cleavage of cysteine S-conjugates derived from glutathione S-conjugates results in the formation of reactive intermediates that may be responsible for the observed nephrotoxicity or nephrocarcinogenicity, or both, of the haloalkenes hexachlorobutadiene, chlorotrifluoroethene, trichloroethene, and tetrachloroethene (1-3). These haloalkenes are conjugated with glutathione by hepatic cytosolic and microsomal glutathione S-transferases; the glutathione S-conjugates and the derived cysteine S-conjugates are toxic and mutagenic. For example, S-(1,2-dichloroviny1)glutathione (DCVG) and S-(1,2-dichlorovinyl)-~cysteine (DCVC) are nephrotoxic in vivo (41, are toxic to renal epithelial cells in vitro ( 5 ) ,and are mutagenic in bacteria (6). The toxicity of DCVG is blocked by inhibitors of y-glutamyltransferase, dipeptidase, and P-lyase, whereas DCVC-induced toxicity and mutagenicity are only influenced b y P-lyase inhibitors (4-6). The chemical structures of the toxic metabolites formed from cysteine S-conjugates by P-lyase have not been fully elucidated, although S-(2chloro-1,1,2-trifluoroethyl)-~-cysteine is metabolized to an acylating agent (7). An a-chlorinated enethiol (1mercapto-l,2-dichloroethene) may be formed by an enzyt Universitat
Wurzburg. *University of Rochester.
matic 0-elimination from DCVC. Although the formation of this thiol may be associated with the cytotoxicity of DCVC (5, 8), this enethiol is not electrophilic, and its formation cannot, therefore, explain the mutagenicity of DCVC and structurally related S-conjugates (6). Hence, we put forward the hypothesis that the enethiol may be a precursor of thioacylating intermediates that may be responsible for both toxicity and mutagenicity. We report herein the identification of the terminal metabolites formed by hydrolysis of the reactive thioacylating intermediate and of the trapped intermediates derived from DCVC and the structurally related S-conjugate S-(1,2,2-trichlorovinyl)-~-cysteine (TCVC) by bacterial &lyase and by N-dodecylpyridoxal bromide (PL-Br) as a model for enzymatic @-eliminationreactions.
Materials and Methods Syntheses. DCVC and TCVC were synthesized as described previously (6). PL-Br was purified and characterized (7) by a modification of a published procedure (9). N,N-Diethyldichlorothioacetamide and N,N-diethylchlorothioacetamide were synthesized from the corresponding acetamides with phosphorus pentasulfide, as described in ref 10. N,N-Diethylchlorothioacetamide: 'H NMR (CDCl,, 400 MHz) 6 1.27 (t,J = 7 Hz, 3 H), 1.38 (t, J = 7 Hz, 3 H), 3.87 (4, J = 7 Hz, 2 H), 3.98 (4, J = 7 Hz, 2 H), 6.25 (s,2 H); I3C NMR (CDCl,, 'H decoupled) 6 10.1, 13.3, 47.4,48.4, 75.1, 189.5. Calcd for C6Hl,SNCl: C, 43.49; H, 7.3; N, 8.45. Found: C, 42.93; H, 7.49; N, 8.13. Purity: 96% by TLC (n-hexanelchloroform 12). Yield: 52%, bp 75 "C (0.5 Abbreviations: BTC,S-2-benzothiazolyl-~-cysteine; DCVC,S-(1,2dichloroviny1)-L-cysteine;TCVC, S-(1,2,2-trichlorovinyl)-~-cysteine; 0lyase, cysteine conjugate P-lyase (EC4.4.1.13);HPLC, high-performance liquid chromatography; GC/MS, gas chromatography/mass spectrometry; PL-Br, N-dodecylpyridoxal bromide; GSH, glutathione.
0893-228x/88/2701-0175$01.50/00 1988 American Chemical Society
Dekant et a1.
176 Chem. Res. Toxicol., Vol. I , No. 3, 1988 Torr). N,N-Diethyldichlorothioacetamide:'H NMR (CDC13,400 MHz) 6 1.28 (t, J = 7.1 Hz, 3 H), 1.39 (t, J = 7.1 Hz, 3 H), 3.90 (q, J = 7.1 Hz, 2 H), 4.00 (9, J = 7.1 Hz, 2 H), 6.85 (s, 1 H). 13C NMR (CDCl,, 'H decoupled) 6 10.1, 13.3,47.4, 48.3, 73.6, 189.5. Calcd for C6HIlSNCl2: C, 36.01; H, 5.54; N, 6.99. Found: C, 36.35; H, 5.79; N, 7.12. Purity: 98% by TLC (n-hexane/chloroform, 1:2). Yield: 65%, bp 72 "C (0.1 Torr). Incubation Conditions. S-Conjugates (5 mM) were incubated with the partially purified bacterial 0-lyase, as described previously (11). After incubation for 20 min at 37 "C, the incubation mixture was acidified to p H 1.5 with 2 N HC1 and extracted with ether. The ether extract was dried over sodium sulfate, and the solvent was removed under reduced pressure. The residues were treated with boron trichloride/methanol ( 1 1 ) and analyzed by GC/MS. Incubation mixtures of S-conjugates (5 mM) with PL-Br (0.5 mM) contained 5 mM dodecyltrimethylammonium bromide and 0.1 mM tetrasodium EDTA in 2 mL of 0.05 M phosphate buffer, pH 8.0. After incubation for 30 min at 37 "C, the reaction mixtures were acidified, extracted with ether, and derivatized for GC/MS analysis, as described above. For trapping experiments, diethylamine (10 mM) or methyl iodide (5 mM) was added 1 min after the start of the incubation. After incubating for 15 min a t 37 "C, the reaction mixtures were extracted twice with 5 mL of chloroform. The organic layers were combined and concentrated under reduced pressure, and samples were analyzed by GC/MS.
Instrumental Analyses. Gas chromatography/mass spectrometry (GC/MS) was performed on a Finnigan MAT 4510 GC/MS-system. DB-5 (30 m; 0.1-Mm film) or DB-1701 (30 m; 1-pm film) fused silica columns (J & W Scientific, Rancho Cordova, CA) were used for gas chromatographic separations. Samples were analyzed by splitless injection (injector temperature, 220 "C) with a linear temperature gradient of 10 "C/min from 40 to 240 "C. The transfer-line temperature was 250 "C. Mass spectrometric data were processed by a Finnigan Incos MAT data system. Gas chromatography with FID detection was performed with identical separation conditions, but with hydrogen as the carrier gas, with a Hewlett-Packard 5790 capillary GC. High-performance liquid chromatography (HPLC) was performed with a Waters (Milford, MA) liquid chromatography system consisting of 2 M6000A pumps, a Model 660 solvent programmer, and a U6K injector coupled to a Hewlett-Packard 1040 diode array detector. Separations were performed with linear gradients (A: H,O, 10% MeOH, acidified to p H 2 with trifluoroacetic acid. B: MeOH) from 10% to 100% solvent B in 40 min. Steel columns (250 X 8 mm) filled with Supelcosil LC-18s (5-wm particle size) were used for analytical separations.
Results Characterization of PL-Br as a Model for Cysteine Conjugate &Lyase. To investigate the ability of P L - B r to simulate the enzymatic (3-elimination reaction catalyzed b y (3-lyase, PL-Br-catalyzed f o r m a t i o n of 2-mercaptobenzothiazole f r o m S-2-benzothiazolyl-~-cysteine (BTC) w a s d e t e r m i n e d b y HPLC. BTC has b e e n developed as substrate f o r the d e t e r m i n a t i o n of (3-lyase activity (12). I n c u b a t i o n of BTC w i t h P L - B r resulted i n a time-dependent loss of BTC and a t i m e - d e p e n d e n t f o r m a t i o n of 2-mercaptobenzothiazole (Figure l), which was identified b y i t s electronic s p e c t r u m ( d a t a not shown). I n c u b a t i o n of the S-conjugate S-benzyl-L-cysteine with P L - B r resulted i n the t i m e - d e p e n d e n t f o r m a t i o n of benzyl m e r c a p t a n , which was identified b y G C ( d a t a not shown).
Selection of Trapping Agents for the Transformation of Intermediates to Stable Products. T o t r a n s f o r m i n t e r m e d i a t e thiols t o s t a b l e thioethers that m a y be analyzed b y G C / M S , benzyl b r o m i d e and m e t h y l iodide were tested as t r a p p i n g agents. The efficiency of these t r a p p i n g a g e n t s was s t u d i e d w i t h S-benzyl-L-cysteine as
the substrate and P L - B r as the catalyst. Benzyl mercaptan f o r m e d b y a (3-elimination c a n b e quantified b y gas chrom a t o g r a p h y as c a n t h e t r a p p e d reaction p r o d u c t s benzyl
L ImM!
M
15
6c1
45
t iminl
Figure 1. Metabolism of S-2-benzothiazolyl-~-cysteine (BTC) by N-dodecylpyridoxal bromide. Consumption of BTC (0) and formation of 2-mercaptobenzothiazole (MBT) (0) were determined by HPLC (see Materials and Methods). Incubation mixtures (2 mL) contained BTC (5 mM), N-dodecylpyridoxal bromide (0.5 mM), and dodecyltrimethylammonium bromide and were incubated a t 37 "C. Data are means *SD from four incubations.
1
I
I126
lkl! ,If,. \ E . , .I, 899S5
1 1
50
70
90
110
13
,
, 150
. ,
170
,
, 190
,
,
210
,
,
230
,
250
.
,
270
m/z
Figure 2. Mass spectrum of methylated chlorine-containing metabolite of S-(1,2-dichlorovinyl)-~-cysteine formed by N-do-
decylpyridoxal bromide and by bacterial cysteine conjugate 0lyase; for incubation conditions, see Materials and Methods. The metabolite was extracted with ether after acidification of the incubation mixture and was esterified by boron trichloride/ methanol. methyl sulfide and benzyl sulfide. Benzyl bromide t r a p p e d less than 1% of the benzyl m e r c a p t a n f o r m e d to yield benzyl sulfide, whereas m e t h y l iodide trapped more than 50% of the benzyl m e r c a p t a n f o r m e d t o yield benzyl m e t h y l sulfide and was, therefore, used i n subsequent experiments. D i e t h y l a m i n e w a s selected as the t r a p p i n g a g e n t for intermediate electrophiles, because i t reacts with alkylating or acylating a g e n t s to f o r m volatile a m i n e s or amides, respectively. In t h e presence of diethylamine u n d e r o u r incubation conditions, t h e acylating a g e n t dichloroacetyl chloride was converted to N,N-diethyldichloroacetamide, which was identified b y i t s r e t e n t i o n t i m e u n d e r various g a s c h r o m a t o g r a p h i c conditions. T h e presence of diethylamine d i d n o t influence t h e PL-Br-catalyzed rate of benzyl m e r c a p t a n f o r m a t i o n f r o m S-benzyl-L-cysteine. Incubation of DCVC and TCVC with PL-Br. Incub a t i o n of D C V C or T C V C w i t h P L - B r resulted i n a t i m e - d e p e n d e n t loss of D C V C and T C V C , as d e t e r m i n e d b y HPLC. After extraction of incubation m i x t u r e s cont a i n i n g D C V C and esterification of t h e p r o d u c t s , t h e presence of t w o chlorine-containing metabolites was indicated by mass spectrometry. T h e mass s p e c t r u m of t h e major metabolite was identical w i t h that of s y n t h e t i c chloroacetic acid m e t h y l ester. T h e mass s p e c t r u m of t h e m i n o r metabolite (Figure 2) showed two large f r a g m e n t s at m / z 75 and 124 (35Cl); m / z 124 represents the molecular ion f o r chlorothionoacetic acid m e t h y l ester. Further s u p p o r t for t h e proposed s t r u c t u r e of chlorothionoacetic acid m e t h y l e s t e r c a n b e derived f r o m t h e f r a g m e n t s at
Chem. Res. Toricol., Vol. 1, No. 3, 1988 177
Thioacylating Metabolites of Cysteine S-Conjugates
R=H, CI
m/z
Figure 3. Mass spectrum of a volatile chlorine-containing meby N-dotabolite formed from S-(1,2-dichlorovinyl)-~-cysteine decylpyridoxal bromide (0.5 mM) in the presence of diethylamine (10 mM). I%
so
m
I .
do
no
130
1%
no
.
,I
1%
2x1
231
250
2m
m/z
Figure 4. Mass spectrum of a volatile chlorine-containing meby N tabolite formed from S-(1,2,2-trichlorovinyl)-~-cysteine dodecylpyridoxal bromide (0.5 mM) in the presence of diethylamine (10 mM).
m/z 93 (M+ - OCHJ and 89 (M+- 35Cl). Under identical incubation conditions, TCVC yielded dichloroacetic acid methyl ester, which was identified by comparison of the recorded mass spectrum (data not shown) with that of synthetic reference compound, as the only chlorine-containing metabolite. When DCVC or TCVC was incubated with PL-Br in the presence of diethylamine, the thioamides N,N-diethylchlorothioacetamide (Figure 3) or N,N-diethylchlorothioacetamide (Figure 4), respectively, were identified by GC/MS as trapped products. Incubation of DCVC and TCVC with Bacterial @-Lyase. T o verify that 0-lyase-catalyzed metabolism of the S-conjugates yields the same products as those obtained by cleavage with PL-Br, DCVC, and TCVC were incubated with 0-lyase from Salmonella typhimurium. The partially purified enzyme (11) exhibited a specific activity of 150 nmol min-l mg-l with BTC as the substrate and metabolized DCVC to chlorothionoacetic acid (Figure 2) and chloroacetic acid, which were identified as their methyl esters by GC/MS. TCVC was metabolized to dichloroacetic acid as the only chlorine-containing metabolite. When attempts were made to trap the reactive intermediates formed by the bacterial @-lyase,the concentration of diethylamine was reduced to 0.5 mM to prevent denaturation of the protein. Under these conditions, traces of N,N-diethylchlorothioacetamidecould be detected by GC/MS in incubations containing DCVC, whereas TCVC did not yield detectable amounts of N,Ndiethyldichlorothioacetamide.
Discussion Several cysteine S-conjugates structurally related to DCVC and TCVC are nephrotoxic in vivo (1-3), are toxic t o rat renal epithelial cells ( 3 ) ,and are mutagenic in the Ames test (8). Their toxicity is dependent on transformation to reactive intermediates by cysteine conjugate @-lyase(1-3). @-Lyaseis a pyridoxal phosphate-dependent
Figure 5. Bioactivation mechanism of S-(1,2-dichlorovinyl)-~cysteine (R = H)and S-(1,2,2-trichlorovinyl)-~-cysteine (R = Cl).
Metabolites identified by GC/MS are underlined. 1, 1mercapto-l,2-dichloroethene; 2, l-mercapto-1,2,2-trichloroethene; 3, chlorothionoacetyl chloride; 4, dichlorothionoacetyl chloride; 5 , chlorothioketene; 6, dichlorothioketene; 7, N,N-diethylchlorothioacetamide; 8, N,N-diethyldichlorothioacetamide; 9, chlorothionoacetic acid; 10, dichlorothionoacetic acid; 11, chloroacetic acid; 12, dichloroacetic acid. enzyme (13-16). Because the chemical mechanism of pyridoxal phosphate-dependent @-eliminationreactions is known (17) and is simulated by PL-Br (9),we characterized the reactivity of this model with cysteine S-conjugates and used it to define the bioactivation mechanism of the S-conjugates DCVC and TCVC. PL-Br catalyzed the formation of 2-mercaptobenzothiazole from S-2-benzothiazolyl-~-cysteine and of benzyl mercaptan from S-benzyl-L-cysteine. These stable thiols have been identified as products of the metabolism of the corresponding S-conjugates by 0-lyase present in bacteria and in subcellular fractions from mammalian liver and kidney ( 4 , 6, 12). These results and the finding that identical end products are formed from DCVC and TCVC by PL-Br and by bacterial 0-lyase indicate that PL-Br simulates the enzymatic reaction. An advantage of the chemical model system over the use of enzymes is its insensitivity to added chemicals, which can therefore be used in sufficiently high concentrations to trap reactive intermediates as stable products suitable for GC/MS analysis. The identified terminal metabolites and trapped intermediates define a bioactivation mechanism for DCVC and TCVC (Figure 5). Initially, a 0-lyase-catalyzed @elimination reaction from DCVC and TCVC yields enethiols 1,2-dichloro-2-merpcaptoethene(1) and 1,1,2-trichloro-2-mercaptoethene (2), respectively. These enethiols could not be trapped with methyl iodide, indicating that they are very short-lived under the experimental conditions used. Benzyl mercaptan, a stable thiol, was transformed efficiently to benzyl methyl sulfide by methyl iodide under identical conditions. The a-chlorinated enethiols may rapidly tautomerize to thioacylating intermediates, whose formation is indicated by the identification of thioacetamides 7 and 8 when the S-conjugates were cleaved in the presence of diethylamine.
Dekant e t al.
178 Chem. Res. Toxicol., Vol. 1, No. 3, 1988
Tautomerism of enethiols 1 and 2 may give thionoacyl chlorides 3 and 4. In fact, thionoacyl chlorides, the tautomers of a-chlorovinyl mercaptans, are not in equilibrium with the thiol configuration; the thionoacyl chlorides are the predominant tautomers, indicating that the vinylic mercaptans tautomerize rapidly to the more stable thionoacyl chlorides (18). Alternatively, a-chlorovinyl mercaptans 1 and 2 may eliminate hydrochloric acid to give thioketenes 5 and 6 . Both thionoacyl chlorides and thioketenes (19) react with nitrogen-containing nucleophiles to give thioamides. Therefore, no discrimination between the two alternative intermediates is possible under the present experimental conditions. Reaction of thionoacyl chlorides 3 and 4 or of thioketenes 5 and 6 with water yields thionoacetic acids 9 and 10, which are slowly hydrolyzed to the corresponding acids 11 and 12. Both chlorothionoacetic acid (9) and chloroacetic acid (11) were identified as metabolites of DCVC, whereas only dichloroacetic acid (12) was identified as a metabolite of TCVC. This difference may be due to a higher rate of hydrolysis of dichlorothioacetic acid caused by the stronger electron-withdrawing effect of the dichloromethyl group. These results define more precisely the structure of the reactive intermediates responsible for S-conjugate-induced toxicity. It has been postulated that the thiols formed from S-conjugates may cause toxicity by reacting with protein thiols to form protein mixed disulfides, thus inhibiting critical enzymatic reactions within the cells (8). The observed weak nucleophilicity and short half-lives of the enethiols argue against a significant contribution of the thiol tautomers as reactive intermediates in S-conjugateinduced toxicity. A significant contribution of the enethiol tautomers in the initiation of S-conjugate toxicity and mutagenicity is also not supported by recent structure/ activity studies (20). S-Conjugates that yield stable thiols, such as pentachlorothiophenol, benzyl mercaptan, or 2mercaptobenzothiazole, upon P-lyase-catalyzed metabolism are not toxic to rat kidney epithelial cells and are not mutagenic, whereas S-conjugates forming unstable achlorinated thiols are highly toxic and mutagenic. Both thioketenes and thionoacyl chlorides, which may be formed as intermediates from S-conjugates by 6-lyase, are potent acylating agents and may contribute to the acute toxicity of S-conjugates by acylation of critical cellular macromolecules, such as proteins and lipids. Because of their electrophilicity they may also interact with DNA to cause the observed mutagenicity of DCVC and TCVC in the Ames test. Experiments designed to establish the chemical nature of reactive intermediates and their role in S-conjugate-induced toxicity are warranted.
Acknowledgment. This research was supported by the Deutsche Forschungsgemeinschaft, SFB 172, Bonn (W.D., K.B., S.V., D.H.) and by NIEHS Grant ES03127 (M. W.A.).
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Dohn, D. R. (1988) “Biosynthesis and metabolism of glutathione S-conjugates to toxic forms”. CRC Crit. Reu. Toxicol. 18,311-341. (2) Elfarra, A. A,, and Anders, M. W. (1984) “Renal processing of glutathione conjugates”. Biochem. Pharmacol. 33, 2729-3732. (3) Lash, L. H., and Anders, M. W. (1986) “Bioactivation and cytotoxicity of amino acid and glutathione S-conjugates”. Comments Toxicol. 1, 87-112. (4) Elfarra, A. A,, Jakobson, I., and Anders, M. W. (1986) “Mechanism of S-(1,2-dichlorovinyl)glutathione-induced nephrotoxicity”. Biochem. Pharmacol. 35, 283-288. (5) Lash, L. H., and Anders, M. W. (1986) “Cytotoxicity of S-(1,2dichloroviny1)glutathione and S-(1,2-dichlorovinyl)-~-cysteine in isolated rat kidney cells”. J . Biol.Chem. 261, 13076-13081. (6) Dekant, W., Vamvakas, S., Berthold, K., Schmidt, S., Wild, D., and Henschler, D. (1986) “Bacterial P-lyase mediated cleavage and mutagenicity of cysteine conjugates derived from the nephrocarcinogenic alkenes trichloroethylene, tetrachloroethylene and hexachlorobutadiene”. Chem.-Biol.Interact. 60,31-45. (7) Dekant, W., Lash, L. H., and Anders, M. W. (1987) “Bioactivation mechanism of the cytotoxic and nephrotoxic Sconjugate S-(2-chloro-1,1,2-trifluoroethyl)-~-cysteine”. Proc. Natl. Acad. S C ~C.S.A. . 84, 1443-1447. (8) Green, T., and Odum, J. (1985) “Structure/activity studies of the nephrotoxic and mutagenic action of cysteine conjugates of chloroand fluoroalkenes”. Chem.-Biol. Interact. 54, 15-31. (9) Kikuchi, J., Sunamoto, J., and Kondo, H. (1985) “Non-enzymatic transamination and 8-elimination of DL-S-benzylcysteine catalyzed by a potent pyridoxal model”. J . Chem. Soc., Perkin Trans. 2 341-345. (10) Bauer, W., and Kuhlein, K. (1984) “Thiocarbonsaure-amide”. In Methoden der organischen Chemie (Houben-Weyl),Bd. XIV, pp 1243, Thieme Verlag, Stuttgart. (11) Dekant, W., Martens, G., Vamvakas, S., Metzler, M., and Henschler, D. (1987) “Bioactivation of tetrachloroethylene: role of glutathione 5‘-transferase-catalyzed conjugation versus cytochrome P-450-dependent phospholipid alkylation”. Drug Metab. Dispos. 15, 702-709. (12) Dohn, D. R., and Anders, M. W. (1982) “Assay of cysteine conjugate $-lyase activity with S-(2-benzothiazolyl)cysteineas the substrate”. Anal. Biochem. 120, 379-386. (13) Stevens, J. L. (1985) “Isolation and characterization of a rat liver enzyme with both cysteine conjugate 8-lyase and kynureninase activity”. J . Biol. Chem. 260, 7945-7950. (14) Stevens, J. L., Robbins, J. D., and Byrd, R. A. (1986) “A purified cysteine conjugate $-lyase from rat kidney cytosol”. J . Biol. Chem. 261, 15529-15537. (15) Stevens, J . L., and Jakoby, W. B. (1983) “Cysteine conjugate 6-lyase”. Mol. Pharmacol. 23, 761-765. (16) Tomisawa, H., Suzuki, S., Ichihara, S., Fukazawa, H., and Tateishi, M. (1984) “Purification and characterization of C-S lyase from Fusobacterium“. J . Biol.Chem. 259, 2588-2593. (17) Walsh, C.(1979) Enzymatic Reaction Mechanisms, pp 777-827, Freeman, San Francisco. (18) Seybold, G. (1975) “Aliphatische ThiocarbonsaurechlorideDarstellung and Eigenschaften”. Angew. Chem. 87,710-711. (19) Scheithauer, S., and Mayer, R. (1979) “Thio and dithiocarboxylic acids and their derivatives”. In Topics in Sulfur Chemistry (Sewing, A., Ed.) Vol. 4, Thieme Verlag, Stuttgart. (20) Vamvakas, S., Berthold, K., Dekant, W., and Henschler, D. (1988) “Bacterial cysteine conjugate &lyase and the metabolism of cysteine S-conjugates: structural requirements for the cleavage of S-conjugates and the formation of reactive intermediates”. Chem.-Biol.Interact. 65, 59-71,
