158 8iochimica et 8iophysica Acta, 957 (1988) 158-163 Elsevier

BBA 33236

Oxidation of 3,4.dihydroxymandelic acid catalyzed by tyrosinase

Francisco Martinez Ortiz a, Jos~ Tudela Serrano b, Jos~ Neptuno Rodriguez L6pez b, Ram6n Varon Castellanos c 9

Jos6 Antonio Lozano Teruel b and Francisco Garcia-C~novas b a Departamento de Qufmica.Fisica. Facultad de Ciencias Quimicas, b Departamento de Bioquimica, Facultad de Medicina,

Untversidad de Murcia, Murcia and c C~tedra de ~u|mica 1, E. U. Politdcnica de AIbacete. Universidad de Castilla-La Mancha (Spain)

(Received 2 June 1988) (Revised manuscript received 3 August 1988)

Key words: Tyrosinase; 3,4-Dihydroxymandel~c acid; (Enzyme kinetics)

Tymsinase usually catalyzes the conversion of monophenols to o-diphenols and the oxidation of o-diphenols to the corresponding quinones. However, when 3,4-dihydroxymandelic acid was provided as the substrate, 3,4-dihydroxybenzaldehyde was produced. These results led to the proposal that tyrosinase catalyzes an unusual oxidative decarboxylation of this substrate (Sugumaran, M. (1986) Biochemistry 25, 4489-4492). However, 3,4-dihydmxybenzaldehyde is also obtained through the oxidation of 3,4-dihydroxymandelic acid by sodium periodate and on a mercury electrode. These results led to the proposal that tyrosinase catalyzes the oxidation of the substrate into o-quinone, which reacts immediately with a molecule of substrate, oxidizing it and through decarboxylation generates an intermediate (qt~inone methide) which transforms into 3,4-dihydmxybenzaldehyde; simultaneously, the original o-quinone is reduced to 3,4-dihydroxymandelic acid.

Introduction

Tyrosinase (EC 1.14.18.1) is a copper protein widely distributed in the phylogenetic scale [1]. It has two different catalytic functions: (a) cresolase, producing the hydroxylation of monophenois (tyrosine) to o-diphenols (dopa) and (b) catecho- lase, oxidizing dopa to o-dopaquinone [2]. The

Abbreviations: topa, 2,4,5-trihydroxyphenylalanine; dopa, 3,4-dihydroxyphenylalanine; DOMA, 3,4-dihydroxymandelic acid; DOMAQ, o-DOMA qulnone; DOBA, 3,4-dihydroxy- benzaldehyde; RPP, reverse pulse polarography.

Correspondence: F. Garcia-C~novas, Departamento de Bioqulmica, Facultad de Medicina, Universidad de Murcia, Murcia, Spain.

products generated by this enzyme, the o-quinones, are very reactive and tend to stabilize by intramo- lecular [3] or intermolecular reactions [4]. This gives, depending on the substrate (o-diphenol) and on the medium conditions, different reaction products [5].

The extent of the reactivity of the o-quinones, and therefore their short half-life, led to the as- signment of different catalytic activities to tyrosinase besides those described previously, such as the hydroxylation of dopa to 3,4,5-trihydroxy- phenylalanine [6], the hydroxylation of dopa to topa [7,8] and the oxidative decarboxylation of DOMA to DOBA, as recently proposed [9].

Previously, we studied the kinetics of the melanin biosynthesis pathway, from dopa to dopachrome [3] and from tyrosine to dopachrome

0167-4838/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

[10], characterizing the chemical steps coupled to the enzymatic process and showing their great importance in the enzyme action. The kinetic study of the oxidation of the different catecholamines such as dopamine, noradrenaline, ot-methyl-dopa and epinine [11-151, allowed us to propose a minor route for the catabolism of catecholamines. In all the papers mentioned above, the generated o-quinones undergo an intramolecular cyclization by means of a 1,4 Michael addition of the amino group of the side-chain on the ring.

In the group of intermolecular reactions real- ized by the product of the action of tyrosinase, we can distinguish those treated with o-dopaquinone at very acidic pH giving topa [16], or in the presence of strong nucleophile such as the thiol group of cysteine giving 5-cysteinyl dopa [14]. Other tyrosinase substrates, such as 4-methyl- catechol, which give o-quinones (not cyclizable) as a product in the presence of amino acids, may undergo nucleophilic attack, giving adducts which are later oxidized to quinones [3].

In most of the above-mentioned cases, the sys- tem reaches steady state after a lag period which is in the time scale of second to minutes, depending on the rate constants involved in the chemical steps coupled to the enzymatic step. However, if the rate constants are high, the lag period will be in the time scale of milliseconds, making possible its overlap with the transient phase of the en- zymatic step.

Taking into account the above, the aim of this paper is to study the oxidation of DOMA by tyrosinase, bringing forward spectrophotometrical and electrochemical experimental evidence which supports the typical mechanism of enzyme action, i.e., the orddation o2 o-diphenol to o-quinone. This o-quinone cannot undergo intramolecular cycli° zation. However, it can oxidize DOMA through an oxidative decarboxylation, giving rise to a quinone methide intermediate that transforms quickly into DOBA.

Materials and Methods

Mushroom tyrosinase (monophenol mono- oxygenase, EC 1.14.18.1, 2230 units/mg) and DOMA were purchased from Sigma. DOBA was obtained from Fluka and sodium metaperiodate

159

was supplied by Merck. Other reagents were of analytical grade.

The enzyme activity was determined spectro- photometrically by the appearance of the product (DOBA) in the reaction medium at 312 nm, in sodium phosphate buffer (pH 6.0) at 25 ° C. Mea- surements were carried out by means of a UV/Vis Perkin Elmer Lambda-3- spectrophotometer inter- faced on-line with a Perkin Elmer computer model 3600 Data-Station. Spectra ~vere recorded on the same instruments, with scanning speeds depend- ing on the reaction rates. Temperature was con- trolled using a Hetofrig circulating bath with a heater/cooler and checked using a Cole-Parmer digital thermometer.

The DC (direct current) and pulse polaro- graphic i / E curves were obtained in an AMEL 471 multipolarograph, using a dropping mercury electrode with mercury flow rate equal to 0.5 mg. s-1. The dropping time was 3 s and the pulse amplitude was 65 ms with a 15 ms sampling time.

Cyclic voltammograms were obtained in an AMEL 563 multipurpose unit, using a hanging mercury drop electrode (METROHM EA 290) with radius equal to 0.06 cm.

In the electrochemical measurements the cell was thermostated and the solutions purged with nitrogen. Potentials were referred to the saturated calomel electrode.

Protein concentration was determined by the modified method of Lowry [17].

Results and Discussion

In order to demonstrate that the reaction of DOMA with mushroom tyrosinase proceeds by a first step of oxidation, in which the corresponding o-quinone is generated, and that the decarboxyl- ation reaction is a chemical reaction enzymatically uncatalyzed, we carried out three types of experi- ment: (a) reaction on a mercury electrode; (b) oxidation by periodate, and (c) oxidation by mushroom tyrosinase.

Reaction on a mercury electrode Fig. 1 shows only one oxidation wave in the

DC polarogram of DOMA. However, in the ex- periments in RPP (reversal pulse polarography) scanned from positive (corresponding to limiting

160

-3 !

- I

0

1

J

/7, i i

I

0

E iv) Fig. 1. DC polarograra (a) and RPP polarogram (b) 0.4 raM DOMA in 70 ram phosphate buffer (pH 7.5) at 25 o C. In RPP the scan starts at +0.2 V. (c) Pulse polarograra of 0.15 mM DOBA under the same conditions. In all cases the cathodic

current is considered negative.

current) to negative potentials, a mixed anodic-cathodic wave and a more cathodic wave were seen to appear. The anodic-cathodic wave is characteristic of a reversible redox couple. Never- theless, the oxidized form is chemically unstable, as revealed by the ratio between cathodic and anodic limiting currents, which is lower than it would be if both species were stable [18]. The more cathodic wave (which is not observed if the scanning is carried out from a potential in which DOMA is not oxidized) corresponds to a final stable product of the oxidation [19]. This product is DOBA, as revealed by comparison with an authentic sample.

The cyclic voltammetric experiments confirm these results. If the rate of scanning is low, only an anodic peak appears; if the rate increases, a cathodic one also appears (Fig. 2). The separation between peaks corresponds to a reversible couple and the displacement of the anodic one when the scan rate increases also indicates the unstability of the oxidized form [20].

Oxidation by periodate Mechanistic considerations and previous stud-

ies of the course of oxidation of catechols with sodium periodate support the concept that catcchols, fike 1,2-glycols, form cyclic periodate ester intermediates which are cleaved in aqueous solutions to o-benzoquinones [21]. In this way, one has a chemical reagent with a similar specific- ity to that of tyrosinase for the oxidation of o-di- phenols.

Oxidation of DOMA by sodium periodate was carried out at pH 6.0 at different [DOMA]/ [NaIO4] ratios from 20 to 2.5. In all cases a compound appeared with Xmax at 312 nm, which was ascribed to DOBA and is in agreement with the spectrum of this compound (Fig. 3A). The calculations of concentrations in those transfor- mations indicate that they occur with a stoichiom- etry 1 : 1 (Fig. 3B). However, one cannot detect an o-quinonic intermediate corresponding to the oxidation by sodium periodate. The high oxidizing power of this o-quinone could cause the dehydro- genation [22] of one molecule of DOMA to DOBA, through a charge-transfer complex leading to oxidative decarboxylation and yielding a quinone methide intermediate [9] as shown in Scheme I:

9

6

d

P ~

< a, 3

O

-3

0 0.1 0.2

E ( V )

Fig, 2. Cyclic voltaramograras of 0.4 mM DOMA in 70 raM phosphate buffer (r~H 7.5). Scan rates were (a) 20 mV. s-U; (b) 50 mV.s-1; (c) 100 raV.s -1 and (d) 200 mV,s -I. Scan starts

at0 V.

161

OH X: COOH DOMA

(fast)

[ °o 1 H H

O ~ f v

Io:, (fast)

I charge - transfer complex

OH

O-DOMA Q

DOMA

quinone methide

(fast )

0

DOBA

Scheme I

Similar results to those shown i "t Fig. 3A are obtained in DC polarography (resul'~s not shown).

The assays carried out on the mercury electrode

are in accordance with Scheme I, since the methide quinone had a very short half-life and would not be detectable as a cathodic peak. Thus, the

1.5

1.0

0.5

. / / " / / /

0 350

. . . . - - . . . ~. . ~

"' ,/'

. , . S - - - - " /

300

c A '"J 'i

I

25O

Mnm)

1.0 A312

0.5

0

B

1 50 |00

[to ] (pM) Fig. 3. (A) Spectrophotometric recording for the oxidation of DOMA with sodium periodate at 25 °C in 30 mM sodium phosphate buffer (pH 6.0). Trace (a) spectra of 0.25 mM DOMA; (b) 0.25 mM DOMA was oxidized with less than one equivalent of 75 #M NalO4. Scan speed 60 rim/rain later recordings did not give variation in the absorbancc). (c) Spectra of 12.5 t~M DOBA. (B) 0.25

mM DOMA was oxidized with concentrations of NaIO4 from 12.5 to 100/~M. A3t2 vs. [NalO4].

2.0

(mi 04

Aa!2

0.2

0

A . 7c~

1,0 2.0 3.0 t (rain)

1.0

I !

0 20 40

162

Fig. 4. (A) Product formation against time for the DOMA oxidation with tyrosinase. Trace (a) absorbance of DOBA accumulation against time followed at ~ = 312 nm during reaction of 20 Fg/ml of tyrosinase with I mM DOMA at 25°C in 10 mM sodium phosphate buffer (pH 6.0). (b) plot of absorbance against time for the same reaction as in (a), but with 25 laM ascorbic acid present. (B) plot of lag period (¢) vs. ascorbic acid. Experiments were performed under the same conditions as in (a), hut with the presence of

ascorbic acid in concentrations of 12-41 t~M.

anodic-cathodic wave in the RPP and the anodic- cathodic peaks in the cyclic voltammetric experi- ments corresponds to the reversible couple:

- 2 e ' - 2H"

D O M A ~ - o - D O M A Q

+ 2e" ÷ 2H*

Scheme II.

Oxidation by tyrosinase When the oxidation of DOMA was achieved by

mushroom tyrosinase, it was again possible to record a peak at 312 nm which was coincident with the maximum previously obtained upon oxidation by periodate (Fig. 3) (results not shown).

When the same assays are performed at a fixed wavelength of ~, = 312 nm, the absorbance in- creases with time and a lag period does not appear in the accumulation of DOBA (Fig. 4A). However, on adding increasing quantities of ascorbic acid an increasing lag period occurs in the accumulation of aldehyde, indicating that the ascorbic acid re- duces an intermediate produced previously to the aldehyde formation. Thus, the sequence of reac- tions must proceed from an oxidation step previ- ous to the decarboxylation, consistent with the resuRs and the mechanisms proposed for sodium periodate and with the mercury electrode. Accord- ing to this, the action of ascorbic acid would be to

reduce o-DOMAQ to DOMA, as shown in the Scheme lII"

Dehydrqscorbic acid

f DOMA

Ouinone nT'ethide

~L ka DOBA

A s c o r ~ acid

It),"

o, "4 -~ o - DOMAQ

Tyrosinose j

DOMA

Scheme !1I.

The lag period can 'be related to the concentra- tion of ascorbic acid. The quantity of material that enters into the system is %. t, where t is the considered time of the enzyme action and v o is the initial rate. This material is distributed according t o

rot = [DOBA] + ~[1] (1)

where I is the possible accumulated intermediates, o-DOMAQ and the qtdi~oi~ .,,..~,~,,.."" " - The ascorbic acid would convert o-DOMAQ back to DOMA and the balance would be:

%t - [ascorbic acid] = [DOBA] + Z[I] (2)

In the absence of ascorbic acid, DOBA is accu-

mulated without a lag period, so that the inter- mediates quickly reach the steady state, fulfilling:

vo = kI[o-DOMAQ][DOMA] = k2[quinone methide] (3)

so we have

v o t - [ascorbic acid I = [DOBA] + ( v o / k l [DOMA])

+(Yolk2) (4)

[DOBA] = Vo{ t - ( 1 / k 1 [DOMA]) - (1/k 2) - [ascorbic acid] }

(5)

If (kI[DOMA]) and k 2 are >> 1, from Eqn. 5 we obtain

[DOBA] ffi o p t - [ascorbic acid] (6)

The accumulation of DOBA in the steady state is linear with time, with t = ,r when [DOBA] -- 0 (see Fig. 4A), therefore, from Eqn. 6 we have:

roe ffi [ascorbic acid], ¢ ffi lag ffi [ascorbic acidl/vo (7)

Eqn. 7 explains the experimental results of Fig. 4B, obtained when the ascorbic acid concentration increased. Furthermore, this reduction has a 1:1 stoichiometry, since the slope of Fig. 4B is equiv- alent to the inverse of the steady-state rate of DOBA production. These data indicate that ascorbic acid reduces a quinone which is an inter- mediate between DOMA and DOBA, regener- ating the former.

The three experimental approaches (oxidation on a mercury electrode, oxidation with sodium periodate and oxidation by tyrosinase) show that the enzyme oxidizes DOMA to its o-quinone, which is very reactive and able to oxidize the substrate, with a simultaneous decarboxylation, generating DOBA (Scheme III).

Since tyrosinases have a very wide substrate- specificity it can be hypothesized that tyrosinases from higher organisms could catalyze this type of reaction. In fact, frog epidermis tyrosinase can indeed catalyze this reaction (results not shown). Keeping this in mind, 3,4-dihydroxymandelic acid which is a product of the catabolism of :,oradrenaline, could be oxidized by tyrosinase in vivo through a minor catabolic route of

163

catecholamines, as has been proposed for dopa- mine, noradrenaline, a-methyldopa and epinine [11-15].

Acknowledgement

This work was partially supported by Grant 85-287 from the Comisi6n Asesora de Investiga- ci6n Cientifica y T~cnica, Spain.

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