Oxygenation of fluorinated tyrosines by mushroom tyrosinase releases fluoride ion

ARCHIVES

OF BIOCHEMISTRY

Vol. 276, No. 1, January,

AND

BIOPHYSICS

pp. 65-69, 1990

Oxygenation of Fluorinated Tyrosines by Mushroom Tyrosinase Releases Fluoride Ion Robert

S. Phillips,*,’

Joel G. Fletcher,*

Robert

L. Von Tersch,*

and Kenneth

L. Kirk?

*Departments of Chemistry and Biochemistry, School of Chemical Sciences, University of Georgia, Athens, Georgia 30602; and TLaboratory of Chemistry, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892

Received May 19, 1989, and in revised form August 28, 1989

The reactions of 2-fluoroand 3-fluoro-L-tyrosine with mushroom tyrosinase have been investigated. Both fluorinated tyrosines are good substrates for tyrovalues similar to those sinase, with V,,, and V,,,/K, for L-tyrosine. Oxygenation of 3-fluorotyrosine produces 5-fluorodopa, as expected. Oxygenation of 2-fluorotyrosine is regioselective, and only 6-fluorodopa was detected by HPLC in reaction mixtures. Oxygenation of both isomers of monofluorotyrosine results in fluoride ion production in the absence of ascorbic acid; however, 2-fluorotyrosine also produces fluoride in the presence of ascorbic acid. These results are consistent with previous studies demonstrating rapid intramolecular cyclization of nascent 6-fluorodopaquinone (M. E. Rice, B. Moghaddan, C. R. Creveling, and K. L. Kirk, 1987, Anal. Chem. 59, 1534-1538), which is competitive with reduction by ascorbate, resulting in elimination of the aromatic fluorine as fluoride ion. 0 1990 Academic

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was reported by Morrison and Cohen to give 6-hydroxydopa (3). In addition, 4-aminophenylalanine is hydroxylated very slowly by tyrosinase from Neurospora (4). However, the reactions of ring-halogenated analogs of tyrosine have not been reported. Fluorine can often be incorporated into biological molecules with interesting results. By virtue of its small size, fluorine can substitute for hydrogen or hydroxy functions in organic compounds with minimal steric constraints (5). However, the high electronegativity of fluorine may have profound effects on the electronic distribution and reactivity of resultant analogs. Previous studies have shown that fluorinated analogs of L-dopa are good substrates for tyrosinase-catalyzed oxidations (6). We have now examined the reactions of the monofluorinated analogs of L-tyrosine, 2-fluoro-L-tyrosine (I), and 3-fluoro-L-tyrosine (II) with mushroom tyrosinase. These results demonstrate that oxygenation of these compounds readily occurs, and that fluoride ion can be produced, especially in the reaction of 2-fluoro-Ltyrosine.

Tyrosinase’ is ubiquitous in nature, catalyzing the oxygenation and oxidation of a variety of phenolic substrates. Although the reactions of alkylated phenols such as p-cresol and p-t-butyl phenol have been studied extensively in vitro [for a recent review of tyrosinase, see (l)], the physiological substrate is generally considered to be L-tyrosine. However, the reactions of relatively few tyrosine derivatives and analogs have been examined. LDopa was reported to undergo hydroxylation at the 5position to give 5-hydroxydopa (2). The hydroxylation of 2,4-dihydroxyphenylalanine by mushroom tyrosinase

Materials

’ To whom correspondence should be addressed. ’ Abbreviations used: TISAB, total ionic strength adjustment buffer; tyrosinase, monophenol, dihydroxyphenylalanine:oxygen oxidoreductase (EC 1.14.18.1); dopa, 3,4-dihydroxyphenylalanine.

Proteins. Mushroom tyrosinase [EC 1.14.18.11 was obtained from Sigma (Lot 26F-9515) as a lyophilized powder. Lyophilized bovine liver catalase and L-amino acid oxidase were also products of Sigma. Tyrosine phenol-lyase was purified from tyrosine-induced cells of Citrobacter freundii (ATCC 29063) as described (7). Protein concentra-

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EXPERIMENTAL

PROCEDURES

65

66 tions were determined by the method of Bradford agent obtained from Pierce Chemical.

PHILLIPS

ET AL.

(B), using the re-

2.Fluoro-L-tyrosine and 3Preparation of fluorinated L-tyrosines. fluoro-L-tyrosine were prepared from m-fluorophenol and o-fluorophenol, respectively, pyruvic acid, and ammonia, using tyrosine phenol-lyase (9). The crude Auorotyrosines precipitated when the reaction mixtures were concentrated. The products were recrystallized from water before use. The 300-MHz iH and 282.4-MHz 19F NMR spectra of the Auorotyrosines, obtained on a Bruker WM-300 spectrometer, were consistent with the expected structures. The isomers of Auorodopa were prepared as previously described (10).

Methods Tyrosinase activity assays. The monooxygenase activity of tyrosinase was measured with a YSI Model 53 oxygen monitor. Reaction mixtures contained 5 pg/ml tyrosinase and various amounts of tyrosine or analogs in air-saturated 0.1 M potassium phosphate, pH 7.0, at 25.O”C. Ascorbic acid (15 mM) was added to suppress the catechol oxidase activity, and catalase (62.5 pg/ml) was added to retard the ascorbate autoxidation, as we have previously used in oxygen electrode assays of dopamine-P-monooxygenase (11, 12). The reactions show a significant lag (as long as 5-10 min) before the steady-state rate is achieved, as has been noted by others for the monooxygenase activity of mushroom tyrosinase (13,14) and mammalian tyrosinase (15). The lag was found to be more pronounced for the fluorinated tyrosines than for tyrosine. Substrate inhibition by the fluorinated tyrosines was observed at concentrations above 1 mM, as is also seen with tyrosine (15). The reported kinetic parameters are calculated based on steady-state rates. The oxygen monitor response was calibrated using L-amino acid oxidase and known amounts of L-phenylalanine. The kinetic parameters were estimated by computer fitting to the hyperbolic form of the Michaelis-Menten equation using a nonlinear least-squares regression program (ENZFITTER, Elsevier BIOSOFT). High-performance liquid chromatography. The reaction mixtures contained 1 mM 2.fluoro- or 3-fluoro-L-tyrosine, 10 mM ascorbic acid, 50 fig/ml catalase, and 50 pg/ml tyrosinase in air-saturated 0.1 M potassium phosphate, pH 7.0. The reaction mixtures were incubated for 30 min to 1.5 h. The products were separated from the proteins by centrifugation through an Amicon MPS-1 centrifugal ultrafiltration filter using a YMP membrane. Products of tyrosinase oxygenation reactions were determined with a Rainin HPLC system in 0.1% trifluoroacetic acid, 5% (v/v) acetonitrile on an Applied Biosystems 2.1 X 220.mm C,, column at 0.2 ml/ min, with detection by absorbance at 270 nm. This wavelength was selected as the optimum for detection of the fluorotyrosine and Auorodopa isomers in a single run, although it is less sensitive for 6-Auorodopa (h,,, = 283 nm). Before injection, samples were diluted 20.fold with 0.1% trifluoroacetic acid, 5% (v/v) acetonitrile. The predilution step was essential for efficient separation of the fluorinated isomers of L-dopa. Quantitative analyses for estimation of product stoichiometries were performed by comparison of integrated areas of peaks in standard mixtures (Fig. 1A) with those of the corresponding peaks in reaction mixtures, using an LDC integrator. Fluoride electrode measurements. Fluoride release upon oxygenation of fluorotyrosines was measured with a Corning Fluoride Electrode using an Ag/AgCl reference electrode. Reactions were performed as described for HPLC experiments, except that 100 pg/ml tyrosinase was added, and reaction mixtures were incubated for 1 h. Samples were diluted with equal volumes of total ionic strength adjustment buffer (TISAB) (16). Standard curves were prepared by addition of known amounts of NaF (10-s to lo-’ M) to a blank reaction mixture, followed by dilution with TISAB. Fluoride ion concentrations in the reaction mixtures were then determined by comparison with the standard curve. Control experiments containing L-tyrosine were also performed, and gave no evidence for fluoride production.

TABLE

I

Kinetic Constants for Tyrosinase Reactions Substrate L-Tyrosine 3-F-L-Tyrosine 2-F-L-Tyrosine

Km” (mM) 0.13 0.016 0.32

V,,, (relative)” 1 0.15 0.71

V,,, /K,,, (relative)

a

1 1.27 0.30

a Kinetic parameters were measured using a polarographic 0, electrode as described under Experimental Procedures. The specific activity for the enzyme preparation was found to be 2.7 pmol/min-mg with 1 mM L-tyrosine as the substrate.

RESULTS Reactions of Fluorinated

Tyrosines

Kinetic parameters for oxygenation of fluorinated tyrosines were determined using a polarographic oxygen electrode assay. The results are summarized in Table I. Both monofluorinated tyrosines are good substrates; however, 3-fluoro-L-tyrosine is a better substrate based on comparison of values of V,,,/&&,,*;ghile 2-fluoro-Ltyrosine exhibits a greater V,,,,, . The K,,, value for L-tyrosine obtained in these experiments is in good agreement with literature values (4). The specific activity with Ltyrosine of the commercial enzyme preparation, 2.7 pmol/min-mg, indicates that the enzyme is only about 3% pure (1). Identification

of Reaction Products

The reaction products were readily identified by HPLC analysis of reaction mixtures containing ascorbic acid, as shown in Fig. 1. 2-Fluorotyrosine and the likely reaction products, 2-fluorodopa and 6-fluorodopa, are readily separated under the described HPLC conditions (Fig. 1A). The reaction of 2-fluorotyrosine gave a single product, identified by its retention time as 6-fluorodopa (Fig. 1B). Furthermore, upon extended incubation, there was no further increase in the 6-fluorodopa, but another product with retention time of 3.7 min was observed (data not shown). Control HPLC experiments with tyrosinase and 6-fluorodopa demonstrated that synthetic 6Auorodopa is not stable under the reaction conditions, but is rapidly converted to a product with a retention time of 3.7 min. In contrast, similar experiments with 2 fluorodopa demonstrated that it is stable under the reaction conditions, and thus it should be detectable if it were formed. Reaction mixtures with 3-fluorotyrosine gave one product, identified as the expected 5-fluorodopa by its retention time (Fig. 1C). No evidence for L-dopa formation from the fluorinated tyrosines was detected in these reactions, although oxygenative defluorination has been observed in other oxygenase reactions (17,lS).

MUSHROOM

TYROSINASE

OXYGENATION

C

Time

(minutes)

FIG. 1. (A) Separation of 2-fluorotyrosine (ta = 11.5 min), 2-fluorodopa (ta = 6.91 min), and 6-fluorodopa (ta = 7.71 min) by HPLC as described under Experimental Procedures. The standard mixture contains 1 mM of each component and was used to estimate substrate and product concentrations in B. (B) Reaction mixture of 1 mM 2Auorotyrosine with mushroom tyrosinase after 30 min of incubation. A new peak, with ta = 7.48 min, is seen, which is assigned to 6-Auorodopa. (C) Reaction mixture of 1 mM 3-fluorotyrosine with mushroom tyrosinase after 1 h of incubation. 3-Fluorotyrosine (ta = 11.14 min) and 5.Auorodopa (ta = 7.81 min) are seen in the reaction (corresponding standards were run but are not shown).

Fluoride Electrode Measurements Reaction mixtures containing ascorbic acid produced negligible fluoride ion from 3-fluorotyrosine during incubation (Table II), while large amounts of fluoride are produced from both isomers of fluorotyrosine if ascorbate is not included (Table II). However, addition of ascorbate does not completely suppress fluoride production in the reaction of 2-fluorotyrosine. Thus, the mechanism of fluoride release must be different for the two fluorinated tyrosines. In the absence of ascorbic acid, melanin is efficiently formed from both of the fluorinated tyrosines as well as from tyrosine, as evidenced by the formation of insoluble dark brown to black precipitates. Thus, these data demonstrate that the oxygenation of fluorinated tyrosines results in release of the aromatic fluorine as fluoride ion. DISCUSSION We have found that both 2-fluoro- and 3-fluoro-L-tyrosines are highly reactive with tyrosinase. The product obtained from hydroxylation of 3-fluoro-L-tyrosine is that anticipated, 5-fluoro-L-dopa, and the stoichiometry was nearly 1:l from HPLC measurements (Fig. 1C). However, hydroxylation of 2-fluoro-L-tyrosine is highly regioselective, producing only a single isomer, identified as 6-fluoro-L-dopa (Fig. 1B). This is the expected effect

67

OF FLUOROTYROSINES

of fluorine on electrophilic aromatic substitution reactions. For example, the partial rate factor for nitration of fluorobenzene at the p-position is 14-fold that for an o-position (19). The relatively low stoichiometry for 6-fluorodopa formation observed in the reaction mixtures containing 2fluorotyrosine (see Fig. 1B) was initially puzzling. Quantitative analysis of HPLC data indicated that the 2-&rorotyrosine was 47% consumed after 1.5 h, but the amount of 6-fluorodopa formed accounted for only 14%. However, when authentic 6-fluorodopa was incubated with tyrosinase under the reaction conditions, it was rapidly converted to another product with a retention time of 3.7 min. The presence of ascorbic acid also does not prevent formation of fluoride ion from 2-fluorotyrosine in the reactions (Table II). Thus, it is clear that 6fluorodopa is not stable under the reaction conditions. In previous studies, Rice et al. found that 6-fluorodopa was oxidized by tyrosinase even more rapidly than dopa (6). Rice et al. also demonstrated that electrochemical oxidation of 6-fluorodopa releases fluoride ion in an irreversible 2-electron process, while electrochemical oxidation of dopa and 2- and 5-fluorodopas proceeds through a 4-electron process (6). Since the electrochemical oxidation experiments can be carefully controlled to produce the quinones without overoxidation, it was proposed that 6-fluorodopaquinone must rapidly undergo an intramolecular cyclization to release fluoride (6). Our results agree with these previous conclusions. The proposed reaction pathway is summarized in Scheme I. Loss of fluoride from the 6-fluorodopaquinone would be expected to lead, after aromatization, to 5,6-dihydroxyindole-2-carboxylic acid, which is generated in the normal melanin biosynthetic pathway by a subsequent 2-electron oxidation (1). These results provide new insights into the mechanism of tyrosinase. In the presence of ascorbate, it is possible to suppress the catechol oxidase activity of tyrosinase and obtain good yields of catecholic products (20). However, it has not been clear from these results if this suppression occurs indirectly via reduction of the qui-

TABLE

II

Release of Fluoride from Fluorinated Tyrosines Substrates’

Ascorbateb

2F-I,-Tyr 2-F-L-Tyr 3-F-L-Tyr 3-F-L-Tyr

+ +

[Fluoride]

(PM)

194.5 424.4 13.2 173.0

a Initial concentrations of fluorinated tyrosines were 0.5 mM. h Ascorbate concentration was 10 mM. +, Reaction mixtures containing ascorbate; -, reaction mixtures from which ascorbate was omitted.

68

PHILLIPS ET AL. F

3-F-L-Tyrosine

g-4

Ku’),

q-q;- oz tyrosmase

F +3 2-F-L-Tyrosine

F

7

tyrosmase (Cd’)

HoH*102-

o++;02e +

3

6-F-L-Dopa

I

aromatization

XDLo2H SCHEME

nones by ascorbate or directly by reduction of the oxidized tyrosinase by ascorbate. Our results with 2-fluorotyrosine suggest that the pathway involves preferential reduction of the binuclear copper center of tyrosinase by the nascent 6-fluorodopa, releasing 6-fluorodopaquinone. If ascorbate reduced the enzyme directly, then 6fluorodopa should be the final product, and this would not produce the observed fluoride ion. Indeed, as mentioned previously, synthetic 6-fluorodopa is rapidly oxidized by tyrosinase even in the presence of ascorbic acid. However, the presence of ascorbate results in a twofold reduction of fluoride formation from 2-fluorotyrosine (Table II). Thus, reduction of the 6-fluorodopaquinone by ascorbate apparently competes with the intramolecular Michael reaction, and a substoichiometric concentration of the 6-fluorodopa accumulates in the steady-state. Since rapid cyclization of the quinone derived from 5fluorodopa was not observed by Rice et al. (6), we observe stoichiometric conversion of 3-fluorotyrosine to 5-fluorodopa in the presence of ascorbate, and the formation of fluoride is strongly suppressed. The release of fluoride which we observe from 3-fluorotyrosine in the absence of ascorbate must be occurring during subsequent melanogenesis. In contrast, fluoride release from 2-fluoro-

I

tyrosine can occur through the intramolecular cyclization as well as through melanogenesis. The production of fluoride ion during biological oxygenation of aryl fluorides has been observed in several other systems. Hydroxylation of 4-fluoro-L-phenylalanine by phenylalanine hydroxylase results in L-tyrosine and fluoride ion (17). Also, hydroxylation of 3,5-difluoro-4-hydroxybenzoic acid catalyzed by para-hydroxybenzoate hydroxylase results in 3-fluorobenzoquinone-5carboxylic acid and fluoride ion (18). However, these examples involved direct oxygenative defluorination of a C-F bond. The results in this paper demonstrate a novel mode for fluoride release, by oxygenation to an electrophilic product which undergoes subsequent nucleophilic substitution. Fluoride release from 2-fluorotyrosine is more efficient than that from 3-fluorotyrosine (Table II). Furthermore, 3-fluorotyrosine is highly toxic, due to its conversion to fluoroacetate in liver metabolism (21), while 2-fluorotyrosine is relatively nontoxic. The release of fluoride ion through the action of tyrosinase on 2-fluorotyrosine in tissues containing high levels of tyrosinase (e.g., melanoma tumors) might lead to selective cytotoxicity. 2-Fluorotyrosine has been submitted for testing for inhibitory activity against melanoma cell lines.

MUSHROOM

TYROSINASE

OXYGENATION

ACKNOWLEDGMENT This research was supported by a grant to R.S.P. from the American Cancer Society (No. CH-382A).

REFERENCES 1. Robb, D. A. (1984) in Copper Proteins and Copper Enzymes (Lontie, R., Ed.), Vol. II, pp. 208240, CRC Press, Boca Raton, FL. 2. Hansson, C., Rorsman, H., and Rosengren, E. (1980) Acta DermatoE. (Stockholm) 60,281-286. 3. Morrison, M. E., and Cohen, G. (1983) Biochemistry 22, 54655467. 4. Toussaint, O., and Lerch, K. (1987) Biochemistry 26,8567-8571. 5. Walsh, C. (1983) Adu. Enzymol. Relat. Areas Mol. Biol. 55, 197289. 6. Rice, M. E., Moghaddam, B., Creveling, C. R., and Kirk, K. L. (1987) Anal. Chem. 59,1534-1538. 7. Phillips, R. S., Ravichandran, K., and Von Tersch, R. L. (1989) Enzyme Microb. Z’echnol. 11,80-83. 8. Bradford, M. (1976) Anal. Biochem. 72,248-254. 9. Nagasawa, T., Utagawa, T., Goto, J., Kim, C.-J., Tani, Y., Kumagai, H., andyamada, H. (1981) Eur. J. Biochem. 117,33-40.

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C. R., and Kirk, K. L. (1985) Biochem. Biophys. Res. Commun. 130,1123-1131. 11. May, S. W., Phillips, R. S., Mueller, P. W., and Herman, H. H. (1981) J. Biol. Chem. 256,2258-2261. 12. May, S. W., Phillips, R. S., Mueller, P. W., and Herman, H. H. (1981) J. Biol. Chem. 256,8470-8475. 13. Duckworth, 1613-1625. 14. Makino, 5735.

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N., and Mason, H. S. (1973) J. Biol. Chem. 248,

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15. Pomerantz, S. H. (1966) J. Biol. Chem. 241, 161-168. 16. Frant, M. S., and Ross, J. W., Jr. (1968) Anal. Chem. 40, 11691170. 17. Kaufman, S. (1961) Biochim. Biophys. Acta 51,619-621. 18. Husain, M., Entsch, B., Ballou, D. P., Massey, V., and Chapman, P. J. (1980) J. Biol. Chem. 255,4189-4197. 19. Sykes, P. (1986) in A Guidebook to Mechanism in Organic Chemistry (Sykes, P., Ed.), 6th ed., p. 160, Wiley, New York. 20. Marumo, K., and Wait, J. H. (1986) Biochim. Biophys. Acta 872, 98-103. 21. Koe, B. K., and Weissman, A. (1967) J. Pharmacol. Exp. Ther.

157,565~573.