formation of cysteine conjugates from dihydroxyphenylalanine and its s-cysteinyl derivatives by...

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Biochimica et Biophysica Acta, 672 (1981) 151--157 © Elsevier/North-Holland Biomedical Press

BBA 29490

FORMATION OF CYSTEINE CONJUGATES FROM DIHYDROXYPHENYLALANINE AND ITS S-CYSTEINYL DERIVATIVES BY PEROXIDASE-CATALYZED OXIDATION

SHOSUKE ITO and KEISUKE FUJITA

Institute for Comprehensive Medical Science, School of Medicine, Fujita-Gakuen University, Toy oake, A ichi 4 70-11 (Japan)

(Received April 24th, 1980) (Revised manuscript received September 5th, 1980)

Key words: Cysteine conjugate; Dihydroxyphenylalanine; S-Cysteinyl derivative; Peroxidase

Summary

Peroxidase-catalyzed oxidation of 3-(3,4-dihydroxyphenyl)alanine (DOPA) and its S-cysteinyl derivatives (cysteinyldopas) in the presence of cysteine was studied by analyzing the products with chromatography on Dowex 50W. Pro- ducts of the oxidation of DOPA were found to be 5-S- and 2-S-cysteinyldopa, 2,5-S,S-dicysteinyldopa, and three unknown compounds A1, B, and C. 5-S- and 2-S-cysteinyldopa were also oxidized as easily as DOPA to give 2,5-S,S-dicysteinyldopa and similar patterns of the unknown compounds. Further oxidation of 2,5~,S-dicysteinyldopa in the presence of cysteine yielded compounds A1, B, and C, whereas in its absence compound B was not formed. From these results coupled with the spectral data, it is suggested that compounds A, and C are the two isomeric dihydrobenzothiazine derivatives of 2,5-S,S-dicysteinyldopa, while compound B is 2,5,6-S,S,S-tricysteinyldopa. These data suggest a possibility that peroxidase may play some role in the formation of cysteinyldopa and related metabolites in vivo.

Introduction

5-S- and 2~-cysteinyldopa arise in melanocytes by the addition of cysteine [1] or its equivalents such as glutathione [2] to dopaquinone which is formed by the oxidation of L-DOPA with tyrosinase. Further oxidation of these care-

Abbreviations: DOPA, 3-(3,4-dihydroxyphenyl)alanlne; 5-S-cysteinyldopa, 3-(5-S-cysteinyl-3,4-dihydroxyphenyl)a]anine; 2-S-cysteinyldopa, 3-(2-S-cysteinyl-3,4-dihydroxyphenyl)alanine; 2,5-S,S-dicysteinyldopa, 3-( 2,5-S,S-dicysteinyl-3,4-dihydroxyphenyl)alanlne.

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chol amino acids leads to the formation of phaeomelanins [3], the yellow to reddish-brown pigments found in feathers and hair [1] and in melanoma [4]. Increased amounts of these catechols and related metabolites have been detected in the urine of patients with metastatic melanoma [ 5,6 ].

Recently, Wick et al. have shown that L-DOPA is selectively toxic to human melanoma cells in vitro [7] and that its analogues exhibit ant i tumor activity against several experimental tumors [8--10]. We have then found that 5-S- cysteinyldopa is much more toxic to a variety of cultured human tumor cells than is L-DOPA [11]. Wick postulated that the mechanism of action may involve oxidation of catechols to o-quinones which subsequently combine with sulfhydryl enzymes [ 10].

Oxidation of DOPA by mushroom tyrosinase in the presence of cysteine gave 5 ~ y s t e i n y l ~ l o p a , 2-S-cysteinyldopa, and 2,5-S~<licysteinyldopa in a ratio of 15 : 3 : 1 [12], which agreed with the in vivo metabolism [6]. Besides tyrosinase, peroxidase [13] and superoxide radical [14] are known to be capable of converting catechols to o-quinones. It was therefore of interest to examine whether DOPA and cysteinyldopas can conjugate with cysteine by the action of peroxidase-HtO2.

Materials and Methods

Materials L-DOPA and horseradish peroxidase (type VI; 265 U/mg) were purchased

from Sigma Chemical Co. 5-S- and 2-S-cysteinyldopa were prepared by the chemical method of Prota et al. [15], separated by chromatography on Dowex 50W as previously described [12] and were purified by recrystallization from water. These crystalline preparations of 5-S- and 2-S-cysteinyldopa gave correct elemental analytical data for C12H16N206" H20 and C12H16N206" 2.5H20, respectively. 2,5~,S-Dicysteinyldopa was enzymically prepared [ 12]. All other chemicals were of the highest purity commercially available. Thin-layer chromatography on cellulose (E. Merck) and paper chromatography were carried out in 1-propanol/1 M HC1 (3 : 2 or 3 : 1, v/v).

Peroxidase-catalyzed oxidation o f DOPA and cysteinyldopas in the presence o f cysteine

50 pmol of either L-DOPA, 5-S-cysteinyldopa, 2-S-cysteinyldopa, or 2,5-S,S- dicysteinyldopa and 100 ~mol L-cysteine were dissolved in 5 ml 0.05 M sodium phosphate buffer (pH 6.8); 2~-cysteinyldopa dissolved incompletely in the buffer, but went into solution during the oxidation. To the mixture incu- bated at 30°C, 2 mg peroxidase was added and then 50 ~mol H202 was added in 5 portions at intervals of 10 min; the addition of H~O2 in 1 portion resulted in the formation of brown pigments. The oxidation was terminated by adding 0.5 ml 6 M HC1 30 min after the final addition ofH202. The reaction mixture was applied on a column (1 × 7 cm) of Dowex 50W-X2 (H ÷ form, 200--400 mesh). After being washed with 20 ml 0.5 M HC1, the column was eluted with 3 M HC1 and fractions of 10 ml were collected and spectrophotometrically analyzed. The products were again chromatographed on a column (1.1 × 18 cm) of Dowex 50W-X2 (200--400 mesh; equilibrated with 2 M HC1; flow rate,

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35 ml/h; 10-ml fractions) and analyzed spectrophotometrically between 240 and 340 nm.

Peroxidase-catalyzed oxidation of 5-S-eysteinyldopa and 2,5-S,S-dicysteinyldopa in the absence of cysteine

50 #mol of either 5~-cysteinyldopa or 2,5-S,S-dicysteinyldopa were dissolved in 5 ml 0.05 M sodium phosphate buffer (pH 6.8). 2 mg of peroxidase and then 5 or 10 ~mol of H202 were added to the mixture. After incubation for 30 rain at 30°C, the oxidation was terminated by adding 0.5 ml 6 M HC1. The reaction products were separated by chromatography under the conditions described above.

Results

Peroxidase.catalyzed oxidation of DOPA and cysteinyldopas in the presence of cysteine

Bayse and Morrison [13] have reported that peroxidases catalyze oxidation of DOPA to dopachrome with consumption of 2 equiv, of H202. When DOPA was oxidized with peroxidase-H202 in the presence of 2 equiv, of cysteine, the dopachrome formation did not take place. The reaction products were separated by chromatography on Dowex 50W using 2 M HC1 as eluent (Fig. 1). Among the products, 2~-cysteinyldopa, 5~-cysteinyldopa, and 2,5-S,S-dicysteinyldopa were identified by comparing chromatographic behavior (column and thin-layer chromatography) and the ultraviolet spectra with those of authentic samples. The yields of these products are summarized in Table I. In addition to these comPounds obtained previously by tyrosinase oxidation of DOPA plus cysteine [12], several unknown compounds including A,, A2, B,

.=

~ 0.5 ,<

- - 5.10~

~.0

f I I I I I I 1

~ 1 I I I I I I 10 20 30 40 50 60 70 80 90

Fraction Number

I

100

F i g . 1. C h r o m a t o g r a p h y o n D o w e x 50W o f p r o d u c t s o f peroxidase-cata lyzed ox ida t ion o f D O P A in the presence o f cyste ine . Absorbances were measured at 2 8 0 n m (¢ ~-) f o r D O P A ; 2 9 3 n m (o o) f o r 2 - S - c y s t e i n y l d o p a ( F r a c t i o n No , 1 3 - - 1 8 ) a n d 5 - S - c y s t e i n y l d o p a ( F r a c t i o n No. 2 0 - - 2 8 ) ; 2 7 3 n m (A A) f o r 2 , 5 - S , S - d i c y s t e i n y l d o p a ; 2 6 3 n m (~ ~) f o r c o m p o u n d B; 2 6 9 n m (s I ) f o r c o m p o u n d C. C o m p o u n d s A 1 a n d A 2 eluted together with 5-S-cyste inyldopa.

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T A B L E I

P R O D U C T S O F P E R O X I D A S E - C A T A L Y Z E D O X I D A T I O N O F D O P A A N D C Y S T E I N Y L D O P A S I N

T H E P R E S E N C E O F C Y S T E I N E

T h e s u b s t r a t e w a s o x i d i z e d w i t h 1 e q u i v , o f H 2 0 2 in the presence o f 2 e q u i v , o f cyste ine except for a ( b e l o w ) in which the substrate w a s o x i d i z e d w i t h 0 .1 e q u i v , o f H 2 0 2 in the absence of eysteine. T h e

yields of the products e x c e p t for 5 - S - c y s t e i n y l d o p a w e r e c a l c u l a t e d f r o m t h e i r m o l a r e x t i n c t i o n c o e f f i -

c i e n t s : 2 - S - c y s t e i n y l d o p a , 3 3 8 0 a t 2 9 3 n m ; 2 , 5 - S , S - d i c y s t e i n y l d o p a , 8 5 6 0 a t 2 7 3 n m . T h o s e o f c o m -

p o u n d s B and C w e r e tentat ively assigned to be 1 3 0 0 0 a t 2 6 3 n m a n d 11 0 0 0 a t 2 6 9 r i m , respectively, for the purpose of compar i son . The yie lds o f 5 - S - c y s t e i n y l d o p a w e r e d e t e r m i n e d b y u s i n g a J E O L J L C - 6 A H

a m i n o a c i d a n a l y z e r .

Substrate Products (%)

1 2 3 4 C o m p . B C o m p . C

D O P A (1 ) 6 0 13 4 6 (7 ) (5 )

5 - S - C y s t e i n y l d o p a (2 ) - - 47 - - 2 0 (6 ) ( 1 3 )

2-S-Cyste inyldopa ( 3 ) - - - - 55 15 (9 ) ( 1 4 )

2 , 5 - S , S - D i c y s t e i n y l d o p a (4 ) - - - - - - 1 5 ( 2 1 ) ( 2 6 )

2 , 5 - S , S - D i c y s t e i n y l d o p a (4 ) a - - - - - - 0 (0 ) ( 4 7 )

and C were produced. In the absence of peroxidase, the formation of cysteinyldopas was not observed.

Peroxidase oxidation of 5~S-cysteinyldopa in the presence of cysteine gave the product of conjugation, 2,5-S,S-dicysteinyldopa, in 20% yield, along with the unknown compounds AI, As, B, and C (Table I). A similar pattern of these compounds was also obtained in comparable yields by the oxidation of 2-S- cysteinyldopa plus cysteine.

Oxidation of 2,5~,S-dicysteinyldopa in the presence of cysteine afforded compounds A1, As, B, and C in yields higher than those from DOPA, 5-S-cysteinyldopa, or 2~-cysteinyldopa. A small amount of brown pigment, possibly phaeomelanin, was also formed.

Reaction pathway in peroxidase-catalyzed oxidation o f DOPA and cysteinyldopas

From the results described above, it was apparent that compounds AI, As, B, and C were derived from 2,5-S,S-dicysteinyldopa. To obtain further infor- mations on the structures of these compounds, 2,5-S,S-dicysteinyldopa was oxidized enzymically with 0.1 equiv, of H202 in the absence of cysteine. The reaction yielded, in addition to a considerable amount of pigment, compounds A1 and C (1 : 4.5 ratio) as the major products. Oxidation with 0.2 equiv, of H202 decreased the yields of these compounds. They were purified by paper chromatography followed by chromatography on Dowex 50W. Compounds A~ and C migrated slightly faster than 2,5-S,S-dicysteinyldopa on a cellulose plate; RF values were 0.29, 0.30, and 0.26 for compounds A1 and C, and 2,5- S,S-dicysteinyldopa, respectively, in the solvent system 1-propanol/1 M HC1, 3 : 2. The ultraviolet spectra of compounds A~ and C closely resembled each other and also had similarities with that of 2,5-S,S-dicysteinyldopa (Fig. 2).

When 5~-cysteinyldopa alone was oxidized with 0.2 equiv, of H202, its benzothiazine derivative was produced in a 72% yield. The compound has been shown to be an immediate product of oxidation of 5-S-cysteinyldopa by tyro-

1 5 5

,.o i , / .............

',)\i " l."i\ l /.:

® .. i/ ~, /I \~\ J

oA

0 I I 1 ~ 240 260 280 300 320 340 360

Wavelength (nm)

F i g . 2. U l t r a v i o l e t a b s o r p t i o n s p e c t r a o f 2 , 5 - S , S - d i c y s t e i n y l d o p a ( ), c o m p o u n d s A 1 ( . . . . . . ), C

( . . . . ), a n d B ( . . . . . . ) in 3 M HC1. A b s o r b a n c e s a t m a x i m a n e a r 2 7 0 n m axe axbi traxi ly f i x e d at 1.0 .

sinase [16]. The ultraviolet spectrum of this compound had two absorption maxima at 250 and 293 nm with a shoulder at 286 nm, while that of 5-S- cysteinyldopa had two absorption maxima at 253 and 293 nm. Thus, the similarity between the spectra of 2,5-S,S-dicysteinyldopa and compound A1 or C closely paralleled that between the spectra of 5-S-cysteinyldopa and its benzothiazine derivative. These spectral data indicate that compounds A1 and C are the two isomeric dihydrobenzothiazine derivatives of 2,5-S,S-dicysteinyldopa {Fig. 3). This assignment was further supported by the NMR spectrum of compound C which exhibited a singlet at ~ 7.58 for an isolated aromatic proton.

OH OH OH OH

R > )' R R -~- R" ~ )' ) R" ~ . ~ ~'S-R

Dopa 5-S-Cysteinyldopa 2-S-Cysteinyldopa 2,5 -S,S -Dicysteinyldopa

OH OH Oil

R-S OH R-S N v C O 2 H R-S COzH

R- " ~ " S - R R - " ~ "~S /

S-R R

Compound B Compound A I or C Comopound C or A I

R=CH2CH(NH2)COzH

F i g . 3 . R e a c t i o n p a t h w a y in t h e p e r o x i d a s e - c a t a l y z e d o x i d a t i o n o f D O P A a n d c y s t e i n y l d o p a s in t h e pres - e n c e o f c y s t e i n e .

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The fact that cysteine was required for the formation of compound B from 2,5-S,S-dicysteinyldopa suggests that the compound may be assigned the structure 2,5,6-S,S,S-tricysteinyldopa (Fig. 3). However, the compound was extrem- ely unstable toward oxidation; it gave during handling a blue tr icochrome-type pigment [1] with a broad absorption maximum at 580 nm in 3 M HC1. There- fore, direct evidence for the structure could not be secured. The structure is unknown for compound A2 which has an absorption maximum at 316 nm in 3 M HC1.

Discussion

So far, three types of enzyme system have been shown to convert catechols to o-quinones, i.e., tyrosinase-O2, peroxidase-H202, and superoxide radical. Oxi- dation of DOPA by tyrosinase in the presence of cysteine is known to give 5-S- and 2-S-cysteinyldopa and 2,5-S,S-dicysteinyldopa [12]. Peroxidase activity has been demonstrated in a variety of cells [17]. It should be noted that, whether peroxidase is involved in the formation of melanin in melanocytes has been a subject of controversy [18]. The superoxide radical is produced in a number of biological processes [19]. Dybing et al. [20] have demonstrated that conjugation of a-methyldopa with glutathione results from the oxidation by cytochrome P-450-generated superoxide radical.

The present results have shown that in the peroxidase-catalyzed oxidation, DOPA combines with cysteine to give 5-S- and 2-S-cysteinyldopa which are then rapidly converted to 2,5-S,S-dicysteinyldopa. Further oxidation of the lat- ter gives rise to the formation of products of cyclization (compounds A~ and C) and of conjugation (compound B). It is notewor thy that 5-S- and 2-S-cysteinyldopa can be oxidized by peroxidase as fast as or even faster than DOPA, while 5-S-cysteinyldopa hardly acts as a substrate for tyrosinase [21]. Presumably, this may be related to the difference in the mechanisms of action of the two enzymes; tyrosinase requires a substrate to be bound to it, thus creating a high degree of steric and electronic preference amoung catechols, while in peroxidase-catalyzed oxidation a catechol acts merely as an electron donor to peroxidase-H202 complexes [22]. In this connection, it should be pointed out that 5~S-cysteinyldopa appears to have a redox potential lower than L-DOPA [21]. L-DOPA analogues as well as 5-S-cysteinyldopa are toxic to a variety of tumor cells including leukemias, melanomas, and neuroblastomas in vitro and in vivo [7--11]. It was postulated that a possible mechanism of action may involve oxidative activation of catechols followed by conjugation with sulfhydryl enzymes [10,11]. If this were the case, enzymes other than tyrosinase should be responsible for the oxidation of catechols in tumor cells, since the toxicity is not restricted to melanoma cells. Peroxidase-H202 could be one of such enzyme systems.

2,5-S,S-Dicysteinyldopa has previously been found to be the major con- stituent of the reflecting spheres of the gar (Lepisosteus) [23]. Metabolites with closely related structures have been isolated as components of iron (III)- binding peptides, adenochromes, which occur in the branchial hearts of Octo- pus vulgaris [24]. T h e compounds adenochromines are the three isomeric amino acids derived from DOPA and 2 molecules of 5-mercaptohistidine. In

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order to account for the exclusive formation of these di-adducts, it may be suggested that 2,5-S,S-dicysteinyldopa and adenochromines in these tissues arise by the action of peroxidase rather than tyrosinase.

Finally, peroxidase-catalyzed oxidation may be a useful tool for the prepara- tion of di-adducts such as adenochromines which can be prepared by tyrosinase oxidation only in very low yields [25] and also for the studies of phaeomelano- genesis, as the reaction can easily be controlled by the amount of H202 em- ployed.

References

1 Thomson, R.H. (1974) Angew. Chem. Internat. Edit. 13, 305--312 2 Agrup, G., Falck, B., Rorsman, H., Rosengren, A.-M. and Rosengren, E. (1977) Acta Dermatovenerol.

(Stockholm) 57,221--222 3 Fattorusso, E., Minale, L., De Stefano, S., Chimino, G. and Nicolaus, R.A. (1966) Gazz. Chim. Ital.

99,969.--992 4 Prota, G., Rorsman, H., Rosengren, A.-M. and Rosengren, E. (1976) Experientia 32,970--971 5 Aubert, Ch., Rosengren, E., Rorsman, H., Rouge, F., Foa, C. and Lipcey, C. (1977) Eur. J. Cancer 13,

1299--1308 6 Prota, G., Rorsman, H., Rosengren, A.-M. and Rosen~cen, E. (1977) Experientia 33,720--721 7 Wick, M.M., Byers, L. and Frei, E., III. (1977) Science 197,468---469 8 Wick, M.M. (1977) Nature 269,512--513 9 Wick, M.M. (1978) Science 199,775--776

10 Wick, M.M. (1979) Cancer Treat Rep. 69,991--997 11 Fujita, K., Ito, S., Inoue, S., Yamamoto, Y., Takeuchi, J., Shamoto, M. and Nagatsu, T. (1980) Cancer

Res. 40, 2543--2546 12 Ito, S. and Prota, G. (1977) Experlentia 33, 1118--1119 13 Bayse, G.S. and Morrison, M. (1971) Biochim. Biophys. Acta 244, 77--84 14 McCord, J.M. and Fridovich, I. (1969) J. Biol. Chem. 244, 6049--6055 15 Prota, G., ScheUilo, G. and Nicolaus, R.A. (1968) Gazz. Chim. Ital. 98,495--510 16 Prota, G., Crescenti, S., Misuraca, G. and Nicolaus, R.A. (1970) Experientia26, 1058--1059 17 Okun, M.R., Donnellan, B. and Patel, R. (1972) Lab. Invest. 27,151--155 18 Shapiro, H.C., Edelstein, L.M., Ravindxa, P.P., Okun, M.R., Blackburn, M., Snyder, M., Brennan, T.

and Wllgram, G. (1979) J. Invest. Dermatol. 72, 191~193 19 Paine, A.J. (1978) Biochem. Pharmacol. 27, 1805--1813 20 Dybing, E., Nelson, S.D., Mitchell, J.R., Sasame, H.A. and Gillette, J.R. (1976) Mol. Pharmacol. 12,

911---920 21 Ito, S., Novellino, E., Chioccara, F., Misuraca, G. and Prota, G. (1980) Experienta 37, 822--823 22 Maehly, A.C. (1955) in Methods in Enzymology, Vol. II (Colowick, S.P. and Kaplan, N.O., eds.), pp.

801--813, Academic Press, New York 23 Ito, S. and Nieol, J.A.C. (1977) Biochem. J. 161, 499--507 24 Ito, S., Nardi, G., Palumbo, A. and Prota, G. (1979) J. Chem. Soc. Perkin, I. 2617--2623 25 Ito, S., Nardi, G., Palumbo, A. and Prota, G. (1979) Experlentia 35, 14--15

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