3-Hydroxykynurenine As a Substrate/Activator for Mushroom Tyrosinase

ABB Archives of Biochemistry and Biophysics 412 (2003) 272–278 www.elsevier.com/locate/yabbi

3-Hydroxykynurenine as a substrate/activator for mushroom tyrosinase

Enrico Sanjust, Gloria Cecchini, Francesca Sollai, Nicoletta Curreli, and Antonio Rescigno*

Dipartimento di Scienze Mediche Internistiche, Cattedra di Chimica Biologica, CSGI, Universita di Cagliari, 09042 Monserrato (CA), Italy

Received 16 December 2002, and in revised form 30 January 2003

Abstract

3-Hydroxykynurenine is a tryptophan metabolite with an o-aminophenol structure. It is both a tyrosinase activator and a substrate, reducing the lag phase, stimulating the monophenolase activity, and being oxidized to xanthommatin. In the early stage of monophenol hydroxylation, catechol accumulation takes place, whereas 3-hydroxykynurenine is substantially unchanged and no signiﬁcant amounts of the o-quinone are produced. These results suggest an activating action of 3-hydroxykynurenine toward o-hydroxylation of monophenols. 3-Hydroxykynurenine could therefore well act as a physiological device to control phenolics metabolism to catechols and quinonoids. Ó 2003 Elsevier Science (USA). All rights reserved.

Keywords: 3-Hydroxykynurenine; Tyrosinase; Polyphenoloxidase; Monophenolase; Activation; Lag time

Tyrosinases, better termed polyphenoloxidases and still is the enzyme of choice as a tool to study the (monophenol, o-diphenol:oxygen oxidoreductase; EC general behavior of polyphenoloxidases. 1.14.18.1), are copper-containing monooxygenases cat- The polyphenoloxidase monophenolase activity is alyzing the o-hydroxylation of monophenols to the characterized by a typical lag time, which has been ex- corresponding catechols (monophenolase activity) and plained by means of kinetic [2] and allosteric models [3]. the oxidation of catechols to the corresponding o-qui- From a kinetic point of view, the lag time is the time nones (diphenolase activity). ‘‘Catecholases’’ lacking required for the met form, unable to react with the monophenolase activity have been considered polyphe- monophenol substrate, to be drawn into the active noloxidases, whose full activity was not seen as a con- deoxy form by the corresponding catechol, arising by sequence of inappropriate assays, and therefore the action of the small amounts of the oxy form usually tyrosinases and catecholases could well be treated as a accompanying the met form. Accordingly, this lag time unicum [1]. These enzymes are widespread in the living is shortened or abolished when a reaction mixture con- world and are known also as phenolases and phenol taining polyphenoloxidase and a monophenol substrate oxidases. These terms are often used without regard to is fed with a catechol; moreover, many reducing agents, any rule, even if tyrosinase is the term usually adopted capable of reducing the met form, shorten or abolish the for animal including human enzymes, and refers to the lag time [4]. Polyphenoloxidases have an active site ‘‘typical’’ substrate, tyrosine. Tyrosine is also a good containing a dicopper center [5], formed by two cupric substrate for the enzyme from the champignon mush- ions, each coordinated by three histidine residues. The room Agaricus bisporus, and this enzyme is therefore resting state of the enzyme (met) can be reduced to the usually called ‘‘mushroom tyrosinase.’’ This has been dicuprous form (deoxy, the only one capable of reacting with molecular oxygen), which can bind dioxygen as a peroxo bridge (l–g2 : g2) between the cupric ions (oxy). * Corresponding author. Fax: +39-070-675-4527. Abundant data on the ﬁne structure of the active site has E-mail address: [email protected] (A. Rescigno). accumulated in the recent years, but the wide variety of

0003-9861/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0003-9861(03)00053-5 E. Sanjust et al. / Archives of Biochemistry and Biophysics 412 (2003) 272–278 273 enzymes studied and the different analytical methods droxykynurenine and tyrosinase seem highly probable. used have given rise to highly contradictory informa- In the present work, we examine 3-hydroxykynurenine tion. For example, widely varying Cu–Cu distances have as a mushroom tyrosinase substrate and/or activator in been calculated for the metpolyphenoloxidase, ranging the light of its peculiar o-aminophenol structure. from 2.8 to 3.6 A [6–8]: obviously, this interionic dis- tance is of the highest importance in the binding mode of substrates, inhibitors, and modulators. The two cu- Materials and methods pric ions show some differences with regard to their coordination geometry [5,9] and have been therefore Chemicals identified as CuA and CuB; no agreement exists with regard to whether CuA [7] or CuB [6] is the site for 3-Hydroxykynurenine was purchased from Sigma– substrate binding. Aldrich (Milan, Italy). As 3-hydroxykynurenine is only Some o-aminophenols have been shown to be rela- slightly water soluble at neutral pH, stock solutions of tively poor substrates for polyphenoloxidase, behaving 3-hydroxykynurenine in 10% dimethyl sulfoxide were as catechols and leading to imino-o-quinones and their freshly prepared just prior to use. 4-Tert-butyl-phenol degradation products [10]. In particular, 3-hydroxy- and 4-tert-butyl-catechol were supplied from Fluka anthranilic acid has drawn our attention [11], as it is a (Milan, Italy). Since 4-tert-butyl-phenol also is practi- tryptophan metabolite for several kinds of organisms. cally insoluble in water, a 5 mM stock solution in 10% 3-Hydroxyanthranilic acid, in addition to being slowly iso-propyl alcohol was prepared. 4-Tert-butyl-1,2-ben- oxidized to the quinoneimine and further to the phe- zoquinone used for HPLC analysis was prepared as noxazinone derivative, cinnabarinic acid, acts as a cat- described in [28]. All other chemicals were of analytical echol analog with respect to its capability of stimulating grade, purchased from Fluka, and used without further the monophenolase activity of mushroom tyrosinase purification. Xanthommatin, the (auto)oxidation prod- and shortening the lag time. In mammals, and most uct of 3-hydroxykynurenine, was prepared by incubat- probably in several other organisms, 3-hydroxyanthra- ing known amounts of 3-hydroxykynurenine with excess nilic acid comes from 3-hydroxykynurenine upon hy- Pleurotus sajor-caju laccase until reaction went to com- drolytic cleavage catalyzed by a kynureninase [12]. pletion. An e value of 10,000 M1 cm1 was found and Therefore, 3-hydroxykynurenine can be well regarded as used for kinetic measurements. a key intermediate in tryptophan metabolism and has been studied with respect to both its chemical and its Enzymes biochemical properties [13–15]. Its (auto)oxidation product is xanthommatin, a polycyclic quinonoid widely Mushroom tyrosinase (43,000 enzyme units (E.U.)/ spread in the living world. Xanthommatin is typical for mg protein) was prepared as described in [29]. Two insect eyes, but is also widespread in other animals; its consecutive chromatographic steps with Bio-Gel Hy- formation and deposition has been related to the de- droxylapatite HTP (Bio-Rad, Milan, Italy) and anion generative process leading to cataractous eye lenses in exchange Macro-Prep DEAE (Bio-Rad) were carried humans [16,17]. 3-Hydroxykynurenine is an effective out. One E.U. caused an increase in A280 of 0.001/min at antioxidant in eye lenses [18] where it s gradually con- pH 6.5 at 25 °C in a 3-mL reaction mix where the final verted to xanthommatin with aging. It has been recog- concentrations were 18 mM potassium phosphate buffer, nized as a carcinogen [19] acting with a metal-mediated 0.3 mM L-tyrosine, and 50–100 units of tyrosinase. The oxidative mechanism. 3-Hydroxykynurenine is also a activity of mushroom tyrosinase on 3-hydroxykynure- neurotoxin [20–23]. Kynurenine-3-hydroxylase, the key nine was monitored by measuring the formation of enzyme for 3-hydroxykynurenine production, has a very xanthommatin. Steady state rate was defined as the low activity within the brain [24], but 3-hydroxykynur- linear part of the product accumulation curve. Lag times enine formed elsewhere easily crosses the blood–brain were estimated by extrapolation of the linear portions of barrier [25], with the aid of the large neutral amino acid the curves to the abscissa or by applying the Pomerantz carrier. The prominent importance of 3-hydroxykynur- equation [30] when appropriate. Tyrosinase from a enine in tryptophan metabolism and its strict biochem- commercial preparation (Fluka) was also used for ical relationship with 3-hydroxyanthranilic acid, which comparative purposes (see Results). This preparation shares with 3-hydroxykynurenine many physiopatho- contained 2200 E.U./mg protein. logical properties, have therefore prompted us to study Laccase was partially purified from a culture medium its interactions with polyphenoloxidase, with regard to of the fungus P. sajor-caju, grown as reported in [31]. contributing to the understanding of its general meta- The culture medium containing the extracellular laccase bolic behavior. In fact, the presence of tyrosinase in was submitted to two consecutive chromatographic human tissues and body fluids has been well ascertained steps with Macro-Prep DEAE and Sephacryl HR-200 [26,27], so physiological interactions between 3-hy- (Pharmacia, Sweden). Laccase activity was measured 274 E. Sanjust et al. / Archives of Biochemistry and Biophysics 412 (2003) 272–278 photometrically by checking absorbance increase at preparation routinely used in most laboratories: after a 525 nm when 50 lM syringaldazine was present in a final native PAGE run, a significant laccase activity was volume of 1 mL of a buffered (50 mM K phosphate, pH readily seen [33]. When the same gels were soaked in a 6) solution of suitably diluted medium. One laccase unit solution of 3-hydroxykynurenine, an intensely yellow was defined as in [32]. spot corresponding to the laccase activity quickly ap- Spectrophotometric measurements were performed peared, whereas a feeble spot corresponding to tyrosi- with an Ultrospec 4000 UV/Vis Spectrophotometer nase became visible only after a few minutes. This (Pharmacia). preliminary observation indicated that 3-hydroxykynurenine is a much better substrate for laccase than for HPLC analysis tyrosinase. That 3-hydroxykynurenine is a good laccase substrate has been confirmed with a partially purified Before performing HPLC analysis, protein-contain- preparation of laccase from P. sajor-caju (Fig. 1); in this ing samples were deproteinized by adding 70% per- way xanthommatin, the main ‘‘final’’ (auto)oxidation chloric acid (about 0.5 M in final concentration) and product of 3-hydroxykynurenine, was prepared to ob- centrifuging at 10,000g for 10 min. The resulting super- tain an e value used for kinetic calculations. From the natant was then filtered through a 0.45-lm pore mem- above considerations, the need to work with a purified brane filter (Millipore). polyphenoloxidase preparation arose. For this reason, Identification of the products of enzymatic activity we have used a purified mushroom tyrosinase, obtained was carried out with a Beckman System Gold apparatus as previously described [11,29], which does not contain equipped with an UV-Vis detector module. The column any laccase trace. used for chromatographic separations was a Chromolith It has to be pointed out that no accidental autoxi- RP-18e ODS-Hypersil (100 4.6 mm i.d.) purchased dation of 3-hydroxykynurenine took place under the from Merck (Darmstadt, Germany). Separations of the experimental conditions adopted for this study. Xan- compounds were achieved with 0.085% phosphoric acid thommatin was obtained also from tyrosinase-catalyzed in water, v/v, (solvent A) and acetonitrile (solvent B) as 3-hydroxykynurenine oxidation, as shown in Fig. 2; mobile phases. 3-hydroxykynurenine is a substrate with a Km of 127 lM Separations were achieved at room temperature by and a Vmax of 367 nmol/min/mg protein. The substance initial gradient elution, 20–50% B in 1 min, followed by shows a relatively high affinity for the enzyme, whereas a second gradient elution, 50–90% B, in 3.5 min. The it is only slowly oxidized in comparison with catechols. detector was set at 280 nm. More details are reported in As expected, no lag time was observed, so 3-hydrox- the corresponding figure legends. ykynurenine could be ascribed to the family of o-aminophenol-type substrates of tyrosinase that behave as Electrophoresis catechol analogs rather than o-aminosubstituted monophenols. This observation is not so obvious as it may Polyacrylamide gel electrophoresis runs were carried appear: 3-aminotyrosine, which is an o-aminophenol out by using a MiniProtean II (Bio-Rad) apparatus and following its instruction manual for the preparation of both running buffers and slab gels. Electrophoretograms were developed with a solution of syringaldazine (1 mM in ethanol). The pink spots on a pale yellow background corresponded to enzyme activity. Alternatively, laccase activity was revealed as very sharp and intensely blue spots when a combination of 4-tert-butyl-catechol and 4-amino-N,N-diethylaniline sulfate was used [33].

Results

To unambiguously assess the substrate nature of 3-hydroxykynurenine, the need for a purified tyrosinase, reasonably void of extraneous enzymatic activities and particularly of laccase, arose. It is common knowledge Fig. 1. Hydroxykynurenine oxidation catalyzed by Pleurotus sajor-caju that o-aminophenols are good substrates for fungal laccase. In a total volume of 1 mL, the reaction mixture included 0.65 laccase units, 100 lM 3-hydroxykynurenine, and 0.1 M K phosphate laccases; most commercial tyrosinase preparations con- buffer, pH 6.5. Spectra were recorded from 300 to 600 nm immediately tain significant laccase amounts [34]. This was shown to after mixing all components in the cuvette (t ¼ 0) and after 5, 8, and be the case in the commercial mushroom tyrosinase 10 min. E. Sanjust et al. / Archives of Biochemistry and Biophysics 412 (2003) 272–278 275

Fig. 2. Absorption spectra of 200 lM 3-hydroxykynurenine in the Fig. 3. Spectrophotometric measurement of monophenolase activity. presence of 16 E.U. tyrosinase. Spectra were recorded from 300 to The reaction mixture in 0.1 M potassium phosphate buffer, pH 6.5, t 600 nm immediately after mixing all components in the cuvette ( ¼ 0) contained 38 E.U. tyrosinase and 0.5 mM 4-tert-butyl-phenol as the and after 5, 10, and 30 min. Other experimental conditions as in Fig. 1. substrate (a). The same experiment performed in the presence of 80 lM 3-hydroxykynurenine (b). and, moreover, strictly resembles the structure of a ‘‘typical’’ substrate, tyrosine, has been described as one of the most potent and specific competitive inhibitors for mushroom tyrosinase [35]. Once that 3-hydroxykynurenine belongs to the o-aminophenol-type tyrosinase substrates was ascertained, its capability of acting as an enzyme activator, as is well known for catechols [1–4] and as was more recently found for 3-hydroxyanthranilic acid, was tested. 4-Tert-butyl-phenol was chosen as the monophenol substrate because it shows a high affinity for the enzyme (Km ¼ 16lM) but a relatively low Vmax (4.5 lmol/min/mg protein) for a monophenol [11]. Moreover, its quinonization product 4-tert-butyl-1,2- benzoquinone is remarkably stable as an o-quinone and cannot cyclize; it simply accumulates in the reaction mixture and can be easily detected. On the contrary, when tyrosine is used as the tyrosinase substrate, the Fig. 4. Influence of 3-hydroxykynurenine concentration on lag time. produced dopaquinone quickly cyclizes. The arising The concentration of 3-hydroxykynurenine ranged from 20 to 240 lM. leukodopachrome is the starting point of a complex Other experimental conditions as in Fig. 3. chemical and enzymatic pathway leading to melanin. Cross-reactions between dopaquinone and 3-hydroxykynurenine also take place and make data interpretation problematic. Fig. 3 shows the dramatic decrease of the lag time in the oxidation of 4-tert-butyl-phenol in the presence of 3-hydroxykynurenine. Fig. 4 shows a plot of (T–t) vs 3-hydroxykynurenine concentrations, where T

is the lag time for 4-tert-butyl-phenol alone and t is the lag time in the presence of 3-hydroxykynurenine. As T is not easily determined due to the very slow oxidation rate of 4-tert-butyl-phenol by mushroom tyrosinase, it has been calculated with the Pomerantz equation [30] and found to be 16.6 min. Consequently, the Km value for 3-hydroxykynurenine as an activator is 21 lM. The activating effect of 3-hydroxykynurenine toward 4-tert- butyl-phenol oxidation by tyrosinase has been studied Fig. 5. Rate modification of monophenolase activity vs 3-hydroxyky- also at different 3-hydroxykynurenine concentrations: nurenine concentration. The Y axis shows the velocity of the reaction the obtained pattern strictly resembles that already as the amount of 4-tert-butyl-1,2-benzoquinone/min formed. Other found for 3-hydroxyanthranilic acid [11] and is shown in experimental conditions as in Fig. 4. 276 E. Sanjust et al. / Archives of Biochemistry and Biophysics 412 (2003) 272–278

tively), in addition to the formation of 4-tert-butyl-catechol and, in a reaction mixture containing 4-tert-butyl- phenol, 3-hydroxykynurenine and tyrosinase (Fig. 6). The presence of some secondary peaks most probably indicates the rise of oxidation products of 3-hydroxyky-

nurenine and/or by-products arising from cross-reactions between remainder reagents and reaction products. Fig. 7 shows the results from an additional experiment where the same reaction mixture was incubated for 20 min; samples were drawn at established times prior to HPLC analysis. This experiment taken together with other separate tests conﬁrmed that in the absence of 3-hydroxykynurenine only a marginal amount of 4-tert-butyl-phenol is converted by mushroom tyrosinase.

Discussion

The study of the system 4-tert-butyl-phenol/4-tert- butyl-catechol/4-tert-butyl-1,2-benzoquinone in the presence of mushroom tyrosinase is particularly in- triguing and represents the starting point for some considerations. 4-Tert-butyl-phenol shows a high affinity for the enzyme but is only slowly oxidized. 4-Tert- Fig. 6. HPLC measurement in time course of 4-tert-butyl-phenol (BP), butyl-catechol is a good substrate, being very quickly 4-tert-butyl-catechol (BC), 4-tert-butyl-1,2-benzoquinone (BQ), and 3- oxidized to 4-tert-butyl-1,2-benzoquinone, but its affin- hydroxykynurenine (HK) concentrations in a reaction mixture con- ity for the tyrosinase active site is comparatively low taining 84 E.U./mL tyrosinase, 0.5 mM 4-tert-butyl-phenol, and 80 lM (Km ¼ 990lM). Therefore, if the assumption of a mu- 3-hydroxykynurenine as the initial concentrations. Chromatograms tual exclusion (i.e., competition) between monophenol obtained after 1 and 6 min of reaction time, showing the decrease of 3-hydroxykynurenine and 4-tert-butyl-phenol and the formation of and catechol substrates from the polyphenoloxidase 4-tert-butyl-catechol and 4-tert-butyl-1,2-benzoquinone. active site is made, relatively high concentrations of 4-tert-butyl-catechol are required to displace 4-tert-butyl-phenol from the bicupric center of metpolyphenoloxidase, thus starting the catalytic cycle. This consideration may be reasonably extended to the case of the couple 4-tert-butyl-phenol/3-hydroxykynurenine. 3-Hydroxykynurenine is not among the best substrates for mushroom tyrosinase but shows a relatively high affinity. The chosen 3-hydroxykynurenine concentration of 80 lM allows for a substantial binding of 4-tert-butyl-phenol to the enzyme, while efficiently elic- iting the oxidation of that monophenol and dramatically shortening the lag time. This latter observation fully meets a Km value for 3-hydroxykynurenine as a tyrosinase activator of 21 lM. A closer inspection of Fig. 7 also shows that a substantial amount of 4-tert-butyl-catechol (up to a con- Fig. 7. Plot of the data obtained by monitoring the experiment of centration of about 70 lM) is formed within the very Fig. 6 over 20 min. Symbols (*) and (}), respectively, indicate the early stage of the reaction, whereas the 3-hydroxyky- concentrations of 4-tert-butyl-phenol and 3-hydroxykynurenine measured after 20 min in two separate experiments carried out in the nurenine concentration is still almost unchanged. By presence of 4-tert-butyl-phenol alone (*) and in the presence of 3- then, almost no 4-tert-butyl-1,2-benzoquinone was de- hydroxykynurenine alone (}). tectable, according to the poor affinity of 4-tert-butyl- catechol for mushroom tyrosinase. By comparing the Km Fig. 5. The concentration decreases of both 4-tert-butyl- values for 4-tert-butyl-catechol and 3-hydroxykynure- phenol and 3-hydroxykynurenine were monitored by nine (as a substrate), the obvious consideration that means of HPLC analysis (after 1 and 6 min, respec- 4-tert-butyl-catechol must be substantially excluded E. Sanjust et al. / Archives of Biochemistry and Biophysics 412 (2003) 272–278 277 from the active site of the enzyme arises, and therefore of Streptomyces antibioticus tyrosinase, FEBS Lett. 442 (1999) only 3-hydroxykynurenine can behave as an activator. 215–220. In other words, 3-hydroxykynurenine efficiently stimu- [10] O. Toussaint, K. Lerch, Catalytic oxidation of 2-aminophenols and ortho-hydroxylation of aromatic amines by tyrosinase, lates o-hydroxylation of phenolic substrates, but their Biochemistry 26 (1987) 8567–8571. further quinonization by polyphenoloxidase is not al- [11] A. Rescigno, E. Sanjust, G. Soddu, A.C. Rinaldi, F. 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