221

Biochimica et Biophysica Acta, 1160 (1992) 221-228

© 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00

BBAPRO 34330

Catalytic oxidation of 2,4,5-trihydroxyphenylalanine by tyrosinase: identification and evolution of intermediates Jos~ Neptuno Rodrlguez-L6pez

a, Marino Bafi6n-Arnao h, Francisco Martinez-Ortiz c, Jos6 Tudela d Manuel Acosta b Ram6n Var6n a and Francisco Garcla-C~inovas d a Departarnento de Qu[rnica-F(sica, E.U. Polit~cnica de Albacete, Universidad de Castilla-La Mancha, Albacete (Spain), b Departamento de Biolog{a Vegetal (Fisiolog(a Vegetal), Facultad de Biolog(a, Universidad de Murcia, Murcia (Spain), c Departarnento de Qu(mica-F(sica, Facultad de Ciencias, Universidad de Murcia, Murcia (Spain) and d Departamento de Bioqu(mica y Biolog(a Molecular, Facultad de Biologla, Universidad de Murcia, Murcia (Spain)

(Received 14 April 1992)

Key words: Dopa; Topa; Tyrosinase; Melanin; Melanin biosynthesis; Melanogenesis The oxidation of 3,4-dihydroxyphenylalanine (dopa) by 0 2 catalyzed by tyrosinase yields 4-(2-carboxy-2-aminoethyl)-l,2-benzoquinone, with its amino group protonated (o-dopaquinone-H+). This evolves non-enzymatically through two branches (cyclization and/or hydroxylation), whose respective operations are determined by pH. The hydroxylation branch of o-dopaquinone-H ÷ only operates significantly at pH ~/200 n M at p H 7.0. This could be d u e to a significant c o n t r i b u t i o n at p H 7.0 of the O H radicals involved in the a n a e r o b i c a u t o x i d a t i o n of topa by H 2 0 2 [21]. F o r these reasons, the a u t o x i d a t i o n of topa was p r e v e n t e d in f u r t h e r exp e r i m e n t s by using a p p r o p r i a t e assay conditions. Oxidation o f topa by tyrosinase and periodate. T h e oxidation of topa by 0 2 catalyzed by tyrosinase at p H 6.0 showed a red c h r o m a t o p h o r e with '~'max 2 7 2 - 4 8 5 n m a n d isosbestic points at 287 a n d 310 n m (Fig.

HO~ io3

1

"~.~'*

3/

'°" I

-

|

TABLE 1

CO0-

L

t .I

I

NH~-,,F.---~.

-o

I. 1

kH+ ]

j

RTQH

HPLC determination for the oxidation of topa in different conditions The assay conditions were the same as described in Fig. 2, except that the assay of oxidation with stoichiometric NalO4 was carried out at two pH values (6.0 and 3.0). Only one substance was detected in all the cases, which has different HPLC- and spectrophotometric properties, depending on the elution pH (7.0 or 3.0). Assay

Autoxidation/ Oxidation by NalO4 Oxidation b y | tyrosinase )

eluted at pH 7.0

eluted at pH 3.0

Rt

Amax

Rt

Araax

(min)

(nm)

(rain)

(nm)

2.9

272-485

8.4

265-390

O~o_NH2 :

2"~H

~-_OA~../~ 0 NH2 ~

RTQ

coo-

DC Scheme II. Pathway for oxidation of topa taking into account the effect of pH. OTQH, o-topaquinone-H +.

225 2B,D). Matrix analysis of this iterative spectrum (data not shown) indicated the appearance of at least two absorbing species kinetically related in solution. When 0.1 mM topa was oxidized by stoichiometric NalO 4 at pH 6.0, the same red chromatophore appeared during the first minute of reaction, its spectrum crossing that of topa at 287 and 310 nm (Fig. 2C,D). The products generated in the oxidation of topa in different conditions were analyzed by HPLC (Table I). From the above experiments, it is evident that topa is oxidized by tyrosinase or by NalO 4 to the same substance, which has a red color under neutral pH and yellow under acid pH. This might be related with the protonation/deprotonation of a chemical group. It has been widely established that the oxidation of o-diphenols and triphenolic compounds by NalO 4 leads to the production of o-quinones [16]. Thus, the direct product of the oxidation of topa by NalO 4 or by tyrosinase must be o-topaquinone-H ÷. The 2-OH group of this quinone is not protonated at pH 6.0, giving rise to a species that is stabilized by resonance, RTQH (Scheme II). At pH 3.0, o-topaquinone-H + is 2-OH protonated and in tautomeric equilibrium with p-topaquinone-H +. However, only one peak was detected at this pH by HPLC (Table I). We shall refer to this yellow chromatophore (a mixture in tautomeric equilibrium) as TTQH (Scheme II). By spectrophotometric measurement at 485 nm of the stoichiometric oxidation of topa with NaIO 4 at pH between 2.5 and 7.5, the pK a value for the 2-OH group in o-topaquinone-H ÷ was found to be 4.5. Thus,

°

A 09

~

0

0.9

0

0.9 A

0 250

A

.

o

i

,~..A

°

B

'

C

300

350 ,x (nm)

T A B L E II

Absorption maxima and molar absorptivity coefficients of dopa and its derivatives Compound

dopa o-dopaquinone-H + topa RTQH TTQH leukodopachrome a dopachrome DHI b melanochrome c

pH 3.0

pH 7.0

Area x

~

Amax

E

(nm)

(M - l cm - l )

(nm)

(M -1 cm -1)

278 280 395 292 270 485

2600 3420 1250 4500 9900 2500

278 280 395 292

2 600 3 420 1250 4 500

265 390 282 310 475 270 296 300 540

10120 1000 9 700 3 600 2 400 3 300

296 310 475 270 296 300 540

a Data obtained from G r a h a m and Jeffs [16]. b Data obtained from Murphy and Schultz [22]. c Data obtained from K/Srner and Pawelek [23].

9700 3600 2400 3300

Fig. 3. Spectropbotometric recordings for the evolution of R T Q H in 75 mM-phosphate buffer, pH 7.0. R T Q H formed by oxidation of topa (0.1 mM) with (A), 0.1 m M NalO4; (B), 3 3 . 3 / ~ g / m l tyrosinase and (C), 33.3 / ~ g / m l tyrosinase removed from the reaction before starting the recordings. Time between recordings, 10 min. Other conditions as described in Materials and Methods and in Results and Discussion.

at pH 7.0 > 4.5 = pK a and pH 3.0 < 4.5 = PKa, RTQH and TTQH were obtained, respectively, in accordance with the above experimental data (Fig. 2 and Table I). The oxidation of topa by non-specific orto-oxidants, such as oxygen (Fig. 2A) or by electrochemical techniques [20], yields p-topaquinone-H ÷, which is proto.I2OH _ nated or deprotonated in its 4-OH group z~PAa p aa v 4 O H x), depending on the pH (Scheme II). By measuring at 485 nm it was possible to determine the Michaelis constant of the enzyme on topa as 1.5 mM at pH 5.0.

Evolution of RTQH at neutral pH The oxidation of topa by stoichiometric NalO 4 at pH 7.0 generated o-topaquinone-H +, which was deprotonated in the 2-OH (RTQH). Under these condi-

226 tions, RTQH was very stable but a slow decay in absorbance at 272 nm, as well as an increase in absorbance at 310 nm, were observed (Fig. 3A). The oxidation of topa by 0 2 catalyzed by a high concentration of tyrosinase at pH 6.0 in the same conditions as Fig. 2B caused the complete oxidation of topa into o-topaquinone-H ÷ to yield RTQH. This was submitted to a pH-jump up to pH 7.0 to study its evolution. In these conditions the evolution of RTQH involved the slow decrease in absorbance at 272 nm, and a generalized increase in absorbance at other wavelengths of the spectrum (Fig. 3B). The same assay conditions were also used to generate RTQH, but the enzyme was removed from the mixture by chromatography on Sephadex G-25. The further evolution of RTQH at pH 7.0 (Fig. 3C) was similar to that observed for RTQH generated with stoichiometric N a l O 4 (Fig. 3A). Matrix analysis of the iterative spectrum for these three experimental conditions (Fig. 3A-C) revealed the presence of at least three absorbing species in solution. The identification from their iterative spectra of the intermediates of the above reaction mixture was difficult. Analysis by HPLC of the corresponding samples showed the significant appearance of RTQH, dopachrome and DHI (Ama x 270-296 nm [22], R t 21.4 min; Table II) in assays with oxidation of topa by NalO 4 or by O2/tyrosinase with further removal of the enzyme. The accumulation of DHI did not occur in assays of

oxidation of topa by 0 2 with tyrosinase present in the reaction mixture during the evolution of RTQH. The oxidation of topa by stoichiometric NalO 4 at pH 7.0 was also followed by cyclic voltammetry (Fig. 4A). At the start of the reaction, a cathodic peak appeared at -0.18 V, indicating the reduction of RTQH into topa, whereas the reverse process originated an anodic peak at -0.14 V. 30 min into the reaction, the cathodic peak at -0.26 V and the anodic peak at - 0.24 V revealed the presence of dopachrome. After 60 min, the appearance of the afiodic peak at + 0.07 V and the cathodic peak at +0.01 was significant, revealing the appearance of DHI. The same results were obtained when topa was oxidized by 0 2 with tyrosinase and further analyzed under the anaerobic conditions used in cyclic voltammetry (Fig. 4B). From the above experiments with spectrophotometry, HPLC and cyclic voltammetry, it is evident that topa is oxidized into o-topaquinone-H + by either NalO 4 or 0 2 with tyrosinase and this yields RTQH by the deprotonation of its 2-OH group. Once RTQH is generated, tyrosinase can be removed from the mixture, and the reaction evolves in a similar way to that of RTQH produced with NaIO4. Thus, RTQH cyclizes slowly into dopachrome, which suffers decarboxylation into DHI, and these compounds are the three species detected by the matrix analysis (Fig. 3A,C), On the other hand, the same process occurs when tyrosinase is not removed from the reaction medium, however, the

0.5 1.5

-1.5 1.3

"