the journal of biological chemistry vol. 261, no. 6, 25 ... · the journal of biological chemistry...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 261, No. 6 , Issue of February 25, pp. 3801-3810,1932 Printed in U. S. A .

Analysis of a Kinetic Model for Melanin Biosynthesis Pathway* (Received for publication, October 11, 1991)

Jose Neptuno Rodriguez-Lopez$, Jose Tudelap, Ramon VaronS, Francisco Garcia-Carmonap, and Francisco Garcia-Canovaspll From the $Departamento de Quimica-Fisica, Escuela Universitaria Politecnica de Albacete, Universidad de Castilla-La Mancha, Albacete and the §Departamento de Bioquimica y Biologiu Molecular, Facultad de Biologiu, Universidad de Murcia, E-30100 Espinardo, Murcia, Spain

The kinetic behavior of the melanin biosynthesis pathway from L-tyrosine up to dopachrome has been studied from experimental and simulation assays. The reaction mechanism proposed is based on a single active site of tyrosinase. The diphenolase and monophenolase activities of tyrosinase involve one single (oxidase) and two overlapped (hydroxylase and oxidase) catalytic cycles, respectively. The stoichiometry of the pathway implies that one molecule of tyrosinase must accomplish two turnovers in the hydroxylase cycle for each one in the oxidase cycle. Furthermore, the steady- state rates of dopachrome production and 0 2 consumption from tyrosine and L-dopa, also fulfill the stoichiometry of the pathway: V&/V& = 1.5 and VgJVk = 1.0, where T represents L-tyrosine, DC represents dopachrome, and L) represents L-dopa. It has been ascer- tained by high performance liquid chromatography that in the steady-state, a quantity of dopa is accumulated ([DlBB) which fulfills the constant ratio [DlBB = R[mlo. Taking this ratio into account, an analytical expression has been deduced for the monophenolase activity of tyrosinase. In this expression kTat = (21 3)k3(K1/K2)R, revealing that kTat is not a true catalytic constant, since it also depends on equilibrium constants and on the experimental R = 0.057. This low value explains the lower catalytic efficiency of tyrosinase on tyrosine than on dopa, (VZ,JKI)/(VE.,/KE) = (2/3)R, since a significant portion of tyrosinase is scavenged from the catalytic turnover as dead-end complex EmetT in the steady-state of the monophenolase activity of tyrosinase.

Melanins are heterogeneous polymers of polyphenolic char- acter and little defined structure with color varying from yellow to black (1). Melanins originate the enzymatic brown- ing in fruits and vegetables as well as the pigmentation of animals. Human deficiency in melanins causes albinism and vitiligo, and great interest has been shown in the involvement of melanins in malignant melanomes, the carcinogenic tumors of the skin. There have also been studies on the possible relationship between neuromelanins and damage of neurons and their selective vulnerability in Parkinson’s disease (2). Melanogenesis starts with the oxidation by O2 of monophenols and/or o-diphenols that yield the corresponding o-quinones, which evolve through coupling nonenzymatic reactions to-

* This paper has been partially supported by a grant from the Comisi6n Interministerial de Ciencia y Tecnologia (Spain), project CICYT ALI89-674. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Y To whom the correspondence should be addressed.

ward the formation of melanins (1). Tyrosinase (monophenol, L-dopa:oxygen oxidoreductase,

EC 1.14.18.1) is a copper enzyme present in microorganisms, plants, and animals. Different tyrosinases obtained from several biological sources have similar structural and functional characteristics (3). The active site of tyrosinase consists of two copper atoms and three states, “met,” “deoxy,” and “oxy” (4-11). Structural models for the active site of these three forms of tyrosinase have been proposed (12-14) and confirmed by transformations into other derivatives (15-17). The monophenolase activity of tyrosinase is coupled to its diphenolase activity and the nonenzymatic reactions from the corresponding o-quinones. These processes can be studied by using a “bottom-up’’ approach, in order to obtain a successful insight into the increasing complexity of the pathway.

The o-quinones suffer nonenzymatic breakdown through polymerization and reaction with a number of reagents such as inorganic ions, reductant agents, thiol and amino compounds, and biological macromolecules (1, 3, 4). The amino group of the side chain of o-dopaquinone is involved in an intramolecular 1,4-addition of Michael into the benzene ring, causing its cyclization into leukodopachrome’ (4). This intermediate is quickly oxidized to dopachrome by another molecule of o-dopaquinone-H+, which is reduced to L-dopa (Scheme I). This process has been kinetically characterized from spectrophotometric data (18) and has also been verified from studies with electron spin resonance (19) and pulse radiolysis (20) techniques. On the other hand, a similar sequence of reactions has been reported for noncyclizable o- quinones, starting with the intermolecular addition of nucleo- philic reagents (21, 22). In fact, the hydroxylation of u- dopaquinone-H’ can also be significant at acid pH (Scheme I), as has been detected (23) and kinetically characterized (24). Thus, the melanogenic o-quinones evolve through cyclization and hydroxylation branches involving regeneration of the respective o-diphenol, but the hydroxylation branch is only significant at acid pH, as has been detected in melano- somes and melanome cells (25). A similar kinetic behavior has been detected and analyzed from a-methyldopa (26, 27) and dopamine (28, 29).

Once the nonenzymatic conversion of o-dopaquinone-H+ up to dopachrome has been clarified (Scheme I), it is possible to study the diphenolase activity of tyrosinase. The structural mechanism of this reaction has been widely studied (4, 13, 30-32), and the three forms of the enzyme considered (Scheme 11). Early kinetic studies into the steady-state of the pathway

’ The abbreviations and trivial names used are: leukodopachrome, 2,3-dihydro-5,6-dihydroxyindole-2-carboxylate; tyrosine, L-tyrosine; dopa, ~-3,4-dihydroxyphenyIalanine; o-dopaquinone, 4-(2-carboxy-2- aminoethyl)-1,2-benzoquinone; dopachrome, 2-carboxy-2,3-dihydro- indole-5,6-quinone; HPLC, high performance liquid chromatography.

3801

3802 Kinetic Mechanism of Tyrosinase

O\

O m C O O - oc

HO -7"- 0 2 E 0' "~~~ T OH

E

Pa HD

SCHEME I. Sequence of reactions of the melanin biosynthesis pathway from tyrosine up to dopachrome.

k * k k' k,

k-2 k.8 k.. TEmet Emet + D EmstD Ersc~ 0 2 e Eorv + D E EorvD

OH QH

SCHEME 11. Reaction mechanism for the diphenolase activity of tyrosinase, coupled to nonenzymatic reactions from o-dopaquinone-H+ up to dopachrome in the melanin biosynthesis pathway.

2 ~ n - D + DC +n+

SCHEME 111. General reaction mechanisms for tyrosinase activities on tyrosine and dopa.

reported an apparent ping-pong mechanism (33,34), a partic- ular case of the true Ter Bi mechanism Uni Uni Bi Uni Ping Pong, with two substrates and two products equal between them (35). This reaction mechanism has been useful for the kinetic characterization of the inhibition of tyrosinase by halides (36) and by slow binding inhibitors such as m-cou- maric acid and mimosine (37, 38). Furthermore, this reaction

E red + 0 2

ka

EoxvT

QH ir: 1 La,,

D

2QH - D + DC + H*

DC

SCHEME IV. Reaction mechanism for the monophenolase activity of tyrosinase, coupled to nonenzymatic reactions from o-dopaquinone-H+ up to dopachrome in the melanin biosynthesis pathway.

mechanism has served as the base for the understanding and kinetic characterization of the suicide inactivation of tyrosinase by o-diphenols such as catechol, L-dopa, and dopamine (39-43). This process is not significant in the time range of a few minutes, as is usual in steady-state kinetic studies, but must be taken into account to prevent biased recordings of enzymatic activity.

The above advances concerning the active site of met, deoxy, and oxy forms of tyrosinase, the nonenzymatic reactions from o-dopaquinone (Scheme I) and the diphenolase activity of tyrosinase (Scheme 11) have not been properly considered in any paper on its monophenolase activity (44- 46).

The structural mechanism for the monophenolase activity of tyrosinase has been widely studied (4,13,30-32) by consid- ering the three forms of the enzyme (Scheme 111). Several kinetic studies of the steady-state of the pathway report the apparent inhibition by an excess of tyrosine (31, 44-46) as well as the lower catalytic efficiency of tyrosinase on monophenols than on o-diphenols (8). Furthermore, the appearance of a lag period has been reported (4, 14, 47-51). This lag period depends on the enzyme and tyrosine concentrations, as has been described from a simplified model of Scheme IV for frog epidermis tyrosinase (52). In this paper (52) neither was the oxygen consumption taken into account, nor the turnover of the enzyme in the pathway established. For this reason no analytical expression was derived for the monophenolase activity involving the three substrates tyrosine, oxygen, and dopa.

The aim of this paper is the quantitative characterization of the kinetic behavior of the monophenolase activity of tyrosinase. The reaction mechanism (Scheme IV) involves all the essential steps of the catalytic cycle (Scheme 111) and is coupled to the nonenzymatic reactions from o-dopaquinone- H' that yield dopachrome and regenerated L-dopa. The reliability of this reaction mechanism and the establishment of the enzymatic turnover in the pathway has been verified by experimental and simulation assays. In this way, a valid analytical expression for the steady-state rate of the monophenolase activity of tyrosinase is derived for the first time in the literature. The corresponding kinetic constants are

Kinetic Mechanism of Tyrosinase

determined and their physical significance discussed. In addition, as regards the relationship between the analytical expressions obtained for the monophenolase and diphenolase activities, the lower catalytic efficiency of the enzyme on monophenols than on diphenols is explained.

EXPERIMENTAL PROCEDURES

Materials-Mushroom tyrosinase (3300 units/mg), tyrosine, and dopa were purchased from Sigma. All other chemicals were of analytical grade and supplied by Merck. Mushroom tyrosinase was purified by the procedure of Duckworth and Coleman (33). Protein concentration was determined by a modified Lowry method (53). The enzyme concentration was calculated taking a value of M , 120,000.

Kinetic Assays-The dopachrome accumulation was spectropho- tometrically followed a t 475 nm ( c = 3600 M-' cm") using a Perkin- Elmer Lambda-2 spectrophotometer interfaced on-line with an AM- STRAD PC2086 computer. The reaction medium was 10 mM sodium phosphate buffer, pH 7.0. To estimate the kinetic parameters on tyrosine, care was taken that recordings reached steady-state cor- rectly, with no significant consumption of substrate, suicide inactivation, or dopachrome breakdown. This aspect was solved by the addition to the reaction medium of a [Dl0 < [D],,/[qo, as well as short assays times. In each assay the fact that the pathway had reached the steady-state was checked by quantifying the [Dl accumulated in the reaction medium. This was done by measuring the absorbance increase at 475 nm produced a t each reaction time after addition of 2 mM NaIO,.

HPLC Assays-Oxidation of tyrosine with tyrosinase in the pres- ence of different amounts of dopa was carried out in a Beckman System Gold liquid chromatography system equipped with a pump model llOB for isocratic elution, a programmable 168-diode array detector (monitoring 250 nm; scanning 200-600 nm), and a system gold IBM PS/2 model 5.1. Samples were introduced via a fixed- volume injector (20 pl) Rheodyne. The compounds were separated in an Ultrasphere-ODS (4.6 X 250 mm; i.d. 5 pm) reversed-phase column and eluted a t flow rate of 1 ml/min with ammonium acetate 50 mM and Na2-EDTA, 5 mM, adjusted at pH 3.0,25 "C. The different peaks were characterized by their absorption spectra. The purity of the peaks was determined by the Real Time Purity Algorithm of the 168- diode array detector. The values of [ T I , [Dl, and [DC] were quantified by interpolation of the peak areas on the linear calibration curves obtained from their standards, that for dopachrome being obtained by the oxidation of dopa with NaI04 in a 1:2 stoichiometry (dopa/ NaI04).

Oxygen Determination-Oxygen consumption was followed by a Hansatech DW oxymeter, based on the Clark electrode. Temperature was controlled at 25 'C using a Haake D1G circulating bath with a heater/cooler and checked using a Cole-Parmer digital thermometer with a precision of kO.1 "C.

Simulated Assays-The kinetic behavior of the reaction mechanism is described by a system of differential equations, whose numerical integration was carried out by using the predictor-corrector algorithm of Adams-Moulton, starting with a fourth order Runge- Kutta method (54). The algorithm was implemented and compiled in TurboBASIC 1.0 on an INVES PC-640A computer (IBM AT-com- patible) with an Intel 80287 arithmetic coprocessor.

The reaction mechanism of the monophenolase activity of tyrosinase (Scheme IV) involves the differential equations as follows.

[&net1 = k-JEmetTI + k-,[EmeDI + k,[EoxyD] - ( k l [ q + kz[Dl)[Emet]

[Eredl = M E m e D I + k-~[Eoxy] - k,[Oz][Ered] [ E m d l = kn[E,,t][D] + kt,[Eoryq - (k--8 + ks)[EmetD] [Zrnetq = kl[q[Emetl - k-JEmetq

[EOXY] = k-4[EoxyTj + k-e[&x$I ks[Ered][Oz] - (k4[rrl + k[Dl + k-d[EoxyI

[EoxyD] k~[Eoxy][D] - (k-6 + k~)[EoxyDl [ B o w r r l = ~4[Eoxy1[q - (k-4 + k~)[EoxyTI 4QHl = kdEmetD1 + k7[Eox,Dl - kapp[QHl

PC1 = kaPp[QM/2 [bl = k--8[EmetDl + k-e[EoxyD] - (kz[E,.t] +

k6[Eox~1)[Dl + (kapp[QW/2)

The initial conditions are [E], = [Ern& + [E,,,],, [ T I = [ T I o , [Dl = [Dl,, [OP]O = 0.26 mM and [E,,t]~/[E,,y]o = 9O:lO. The [ T I was considered constant throughout the simulations, in accordance with experimental data.

The mechanism of the diphenolase activity of tyrosinase (Scheme 11) is described by the following system of differential equations.

The initial conditions are [E], = [Ern& + [E,,,]o, [Dl = [Dlo, [02]0 = 0.26 mM and [E,,t]o/[E,,y]o = 9O:lO. The [Dl was considered constant throughout the simulations, in accordance with experimental data.

Assignment of Constants-The values of the equilibrium and rate constants of the model were assigned by taking into account the kinetic analysis of the mechanisms as well as the experimentally determined kinetic parameters. By nonlinear regression of the Vo values versus [Dl, and [ g o , the parameters Kg, VgaX, KZ, and Vzex were determined. The fitting of the integrated Michaelis equation for oxygen consumption gave K?. From VE., the catalytic constant k3 was determined. For the Michaelis constants of Eoxy on tyrosine and dopa the magnitude order was taken from the literature (8). kapp corresponds to the processes of protonation-deprotonation of o-dopaquinone-H+, cyclization, and the further oxidation-reduction, which yield the formation of dopachrome and the regeneration of dopa in the medium (18). Thus, the set of values for the rate constants of the reaction mechanisms of tyrosinase was obtained (Table I).

RESULTS AND DISCUSSION

Stoichiometry of the Pathway-The melanin biosynthesis pathway from tyrosine to dopachrome consist of enzymatic reactions of tyrosinase on tyrosine and on dopa yielding o- dopaquinone-H', which evolves nonenzymatically toward dopachrome (Scheme I). The overall stoichiometry of the pathway involves, therefore, the catalytic turnover of the enzymatic reactions as well as further nonenzymatic steps. Thus, the conversion of tyrosine up to dopachrome is defined by the following mass balance (Scheme IV);

7' + E,,, + 2H+ 4 D + E,,, + H 2 0 D + Erne, 4 QH + Edeory + 2H'

0 2 + Edeoxy 4 E,,, T + E,,, + 2H' 4 D + E,,, + H 2 0

D + E,,, 4 QH + Edeory + 2H'

0 2 + Edeoxy 4 E,,, 2 Q H * D + D C + H +

D + Eo,, + 4H' 4 QH + E,.t + 2H' + 2H20 D + E m e t -+ QH + Edeory + 2H'

0 2 + &eoxy 4 E,,, 2 Q H + D + D C + H +

27' + 30, "+ 2DC + 2H+ + 4H20

whereas the mass balance for the conversion of dopa up to dopachrome (Scheme 11) is as follows.

D + Erne, 4 QH + Edeory + 2H+ 0 2 + Edeoxy 4 E,,,

D + Eoxy + 4H+ 4 QH + E,,, + 2H' + 2H,O (2) 2QH 4 D + DC + H'

D + 0-8 4 U C + H' + 2H20


TABLE I Values of the rate constants used for simulation of the melanin

biosynthesis pathway, taking into consideration the reaction mechanism of tyrosinase

Michaelis Binding constants

Transformation constants constants

K I = 1.9 X lo-' M kl = 5.5 x lo7 M-' s-' kZi = lo's-' k-l = 1.0 X 104 s-1 ks = 10" s-'

KE = 1.4 X M k, = 7.3 X lo7 M-I sC1 ki = lo3 s-' k-, = 1.0 X lo' s-' k,, = 0.41 s-'

KZxy = 1.0 X M k4 = 2.0 X 10' M-I s-' k-' = 1.0 X lo3 s-'

~t~~ = 1.2 x 10-6 M k6 = 1.6 x 109 M-1 s-1

K? = 1.9 x M ks = 5.5 x 10' M - I s-1

k-6 = 1.0 X 103 8-1

k-s = 1.0 X 103 s-l

The melanogenesis pathway from tyrosine starts with the monophenolase activity of tyrosinase (Scheme IV), which consist of two catalytic cycles overlapping through three com- mon intermediates. The stoichiometry of the pathway (balance l ) implies that one molecule of tyrosinase must accomplish two turnovers in the hydroxylase cycle for each one in the oxidase cycle (Scheme IV). Thus, tyrosinase operates three times, twice and once through the steps controlled by k3, kg, and k7, respectively. Therefore, in the steady-state of the pathway, the following rate ratios must be fulfilled.

k,[Eme,Dl = (3/2)kdEoxsrl = %[&&I ( 3 )

The reliability of these ratios has been verified from simulation assays (Fig. 1, A-C) in the final steady-state of the pathway (from 600 s). At a shorter time range (2.0 s) there is an early steady-state restricted to the hydroxylase cycle (Fig. 1A) with k3[E,,tD]/k5[E,,,~ = 1.0, which evolves toward the overall steady-state of the pathway with k3[E,, t~]/k5[&,, ,~ = 1.5. On the other hand, the melanogenesis pathway from dopa starts with the diphenolase activity of tyrosinase (Scheme 11), defined only by the oxidase cycle and one single turnover (balance 2). In the steady-state of the pathway, therefore, the rate ratio is verified as follows,

k,[EmetDl = k 7 [ E o x y D ] , (4)

in accordance with simulation assays (Fig. 1D). The rate ratios (Equations 3 and 4) are useful in the derivation of the steady-state rate equations for the catalytic activities of tyrosinase (see "Appendix").

The stoichiometry of the melanin biosynthesis pathway from tyrosine (balance 1) and from dopa (balance 2) implies thatV$/Vgc = 1.5 and V&/V& = 1.0, respectively, values also obtained in experimental (Fig. 2 A ) and simulation (Fig. 2B) assays. These results (Figs. 1 and 2) support the reliability of the reaction mechanisms proposed for the monophenolase (Scheme IV) and diphenolase (Scheme 11) activities of tyrosinase, involved in the pathway under study (Scheme I).

Accumulation of [Dl,-The operation of the melanogenesis pathway from tyrosine (Scheme IV) can be monitored at 475 nm, and shows a transient phase that evolves toward the overall steady-state of the pathway, with linear production of dopachrome (Fig. 3A). This process can also be followed in a discontinuous way by titration of the [Dl accumulated in the assay medium with NaI04. Thus, a sigmoid pattern of [Dl uersus time, whose final plateau corresponds to the level of [Dl,, is obtained (Fig. 3A), simultaneously with the linear formation of dopachrome. The [Dl,, is not dependent on [E], (Fig. 3B), whereas it is proportional to [ T I o (Fig. 3B),

[Dlss = R [ r l s S E R[%, (5)

since the consumption of tyrosine is negligible during the assay time (Fig. 3A). The same behavior and dependencies were obtained from simulation assays (results not shown).

The transient phase of the pathway (Fig. 3A) involves the regeneration of dopa in the nonenzymatic reactions from o- dopaquinone-H+, as well as the reported competition of tyrosine and dopa on the Emet and E,,, forms (14) (Scheme IV), which is consistent with the nondependence of [Dlss on [El0 (Fig. 3B). Thus, the increasing levels of [Dl remove Emet from

~

0 600 1200 t (SI

0 600 1200 t(s)

0 50 100 t (SI

FIG. 1. Evolution with time of several rate ratios of the catalytic cycle. A-C, monophenolase activity (Scheme IV), with 0.2 mM tyrosine, 0.26 mM O,, and 0.1 PM tyrosinase. D, diphenolase activity (Scheme II), with 0.2 mM dopa, 0.26 mM 0 2 , and 0.1 y M tyrosinase.

Kinetic Mechanism of Tyrosinase 3805

2

V 0 > \ N

P I

2

0 0 > \ N

9 1

I

6

(O)CDl, (mM) FIG. 2. A, experimental results of relation between steady-state of

02 consumption and dopachrome formation: 0, dependence on [Dlo, with [E10 = 1.6 nM; A, dependence on [qo, with [ E ] , = 5.0 nM; B, relation between steady-state rates of 0, consumption and dopachrome formation obtained by simulation: 0, dependence on [Dl0, with [E]" = 0.1 PM; A, dependence on [qo, with [Elo = 0.1 FM.

the dead-end complex EmetT, yielding E,,,&, which is involved in the oxidase cycle (Scheme IV). This increases the operative [E] as well as the formation of o-dopaquinone-H+ and dopachrome up to the attainment of the overall steady-state of the pathway (Fig. 3A) . Therefore, the mon'ophenolase activity of tyrosinase (Scheme IV) starts with the single operation of the hydroxylase cycle and evolves toward the steady-state, with two turnovers in the hydroxylase cycle for each one in the oxidase cycle, as well as a net decrease in the early [EmetT] due to the regeneration of dopa in the nonenzymatic reactions from o-dopaquinone-H+. For this reason, at higher values of [7'l0 there is an increase in the early [EmetTJ, and greater values of [Dlss are required for the attainment of the steady- state of the pathway (Fig. 3 3 ) .

The removal of Emet from the dead-end complex EmetT could be carried out by any diphenol present in the assay medium such as dopa or leukodopachrome, according to their respective levels in the steady-state of the pathway (Scheme IV). The quantity of [Dl,, has been experimentally determined (Fig. 3, A and B ) , whereas leukodopachrome has not been detected in the reaction medium (18, 24). The value of [L],, can be calculated from the following.

[LI = k1o[Q1 - ~ I I [ Q W [ L I = 0 (6 )

Therefore (24),

I / I

0.1 1 / I

0 1.0 2 .o ( 0 ) [TI, (mM)

FIG. 3. A, experimental results of time course of the accumulation of dopachrome ( a ) and dopa ( b ) in the monophenolase activity of tyrosinase. Reagents: 0.2 mM tyrosine, 0.26 mM 0 2 , and 5.0 nM tyrosinase; B, corresponding values of [Dl,, determined by HPLC assays: 0, dependence on [TI,, with ( E ] , = 5.0 nM; A, dependence on [Elo with [ T I o = 1 mM.

since k-9[H'] s k,, at pH 7.0, and where k, = 0.41/s (18) and k,, > lo9 M" s-' (55). Thus, the contribution of leukodopachrome (14) perhaps could be significant early in the reaction, but is negligible at the steady-state of the pathway, due to the low value of [L],, (Equation 7).

Induction Periods in the Production of Dopachrome from Tyrosine-The induction period observed in the production of dopachrome from tyrosine (Fig. 3 A ) is a lag time that increases when [El0 is decreased or [ T I 0 is increased (52). Several tentative explanations for these properties have been proposed by different authors. Thus, a model has been proposed (48) with two consecutive enzymatic reactions in different catalytic sites and which predicts an exponential accumulation of [Dl uersus t instead of the experimental sigmoid pattern (Fig. 3 A ) . A similar model (33) proposes the direct conversion of tyrosine into dopa and the further oxidation of dopa, with no net accumulation of dopa in the medium. Other authors have suggested the slow generation of dopa from tyrosine through nonenzymatic reactions or the strong binding of dopa to tyrosinase (50), as well as the action of dopa as positive effector on one allosteric site of tyrosinase (51), with no supporting evidence from structural or kinetic studies in either case. Furthermore the lag period (Fig. 3 A ) has been attributed to the conversion of Eoxy into E,,, (14), but this process is fast and occurs in the milliseconds range (8).

All the interactions between tyrosinase, tyrosine, and dopa in the monophenolase activity of tyrosinase take place at the binuclear copper site of tyrosinase, without allosteric phenom- ena (14). The lag period, therefore, involves (Scheme IV) the regeneration of dopa in the nonenzymatic reactions from o- dopaquinone-H', as well as the removal of E,,, from the dead- end complex E,,,T to incorporate E,,,D into the oxidase cycle (52).

The above interpretation suggests that the lag period of the monophenolase activity of tyrosinase on tyrosine should be shortened by the addition of dopa at the start of the reaction, due to the lower time required for the attainment of the corresponding [D],,. In fact, there are three possible initial conditions: [Dl0 < [Dl,, and [Dl0 > [Dlss lead to the lag and


burst induction period, respectively, whereas the condition [Dl0 = [Dls3 does not originate induction period. These three cases have been obtained in experimental (Fig. 4) and in simulation (Fig. 5 ) assays. The parallel straight lines show that the production of dopachrome evolves toward the same steady-state of the pathway (Figs. 4 and 5), only determined by[E]o and [q0. HPLC has been applied to experimental assays (Fig. 4) with the three initial conditions (Fig. 6, A-F), obtaining the same value of R (0.057), which is also equivalent to that calculated from simulation assays (Fig. 5 ) . Further- more, the rate ratios (Equation 3) are only accomplished in the steady-state of the pathway. Note that when no dopa is added to the medium the consumption of tyrosine may become significant, making the measurements for kinetic studies dif- ficult (Fig. 6, A and B ) . However, when the reaction starts with [q0 and [Dl0, [!l‘ls8 [q0 (Fig. 6, C-F) and, therefore, this procedure is recommendable for kinetic studies on the pathway (Scheme IV). The [Dl0 chosen can be near to [Dlss determined by titration with NaI04 or by HPLC.

Therefore, an inappropriate experimental design (44-46) has led to misleading conclusions about the monophenolase activity of tyrosinase. There are several points which arise from these studies and which should be commented on:

(a) The appearance and disappearance of a lag period at different pH values may be due to the use of a slow discontinuous method for the measurement of the monophenolase activity. This procedure is not suitable for the determination of transient phase parameters, such as the lag period.

( b ) The observed inhibition of tyrosinase by an excess of tyrosine is possibly due to the use of the same assay time for different [TI,. Thus, at high [ T I o , the lag period increases and “apparent slopes” can be determined, which are lower than the true steady-state rates.

(c) The use of high concentration of ascorbic acid causes the reduction of o-dopaquinone-H+ to dopa, whose continuous accumulation prevents the attainment of steady-state in the pathway (Scheme I). Furthermore, this reagent can originate

“0 lo t(min) 20

FIG. 4. Experimental results of time course of dopachrome accumulation, with several values of [Dl0, (pM): a, 0; b, 6; c, 11; d, 17; e, 22. In all cases 0.2 mM tyrosine, 0.26 mM 02, and 5.0 nM tyrosinase was used.

0 t (m id

15

FIG. 5. Simulation results of time course of dopachrome accumulation, with several values of [Dl0 (pM): a, 0; b, 11; c, 22. In all cases 0.2 mM tyrosine, 0.26 mM 02, and 0.1 p M tyrosinase was used.

6 10 14

D

6 10 14 Retention time (min)

FIG. 6. HPLC assays of hydroxylation of tyrosine catalyzed by tyrosinase, with several values of [Dl0 ( p ~ ) : A-B, 0; C-D, 11; and E-F, 22. Reagents: 0.2 mM tyrosine, 0.26 mM 02, and 5.0 nM tyrosinase. 1, tyrosine; 2, dopa; and 3, dopachrome.

the reduction of Emet into Edeoxy, which modifies the enzymatic turnover.

( d ) The proposal of a model for the monophenolase activity of tyrosinase, with pH-dependent interconvertible forms and one allosteric site for the inhibition by an excess of tyrosinase does not take into account the occurrence of Emet, Edeoxy, and Enry forms and is not supported by any structural evidence.

Kinetic Analysis-The experimental and simulation assays carried out in the above sections support the reliability of the proposed reaction mechanism (Scheme IV) and permit the turnover of the enzyme in the pathway to be established. The constancy of R is confirmed and its value for this enzyme calculated (Table 11). Therefore, it becomes possible to derive the corresponding rate equation for the steady-state of the pathway, which enables its quantitative characterization.

The kinetic analysis of the monophenolase activity of tyrosinase has not been properly accomplished, since conven- tional reaction mechanisms with only one single cycle have been proposed, such as bisubstrate ping-pong and ordered mechanisms (49). Furthermore, kinetic analysis of the pathway from tyrosine up to dopachrome has been attempted without taking into consideration the regeneration of dopa in the nonenzymatic reactions from o-dopaquinone-HC (14).

In the “Appendix” are detailed the kinetic analyses of the monophenolase (Scheme IV) and of the diphenolase (Scheme 11) activities of tyrosinase, taking into account the turnover of the enzyme in this pathway and the contribution of the nonenzymatic reactions from o-dopaquinone-H+ to the melanogenesis pathway. The steady-state rate V& (Equation 2A) is dependent on [Elo and on all the substrates involves in the reaction [ T I o , [Dlsb, and [OZlO (Scheme IV). Moreover, at saturating conditions of [O2lO and according to Equation 5 , Equation 2A is simplified to Equation 5A. However, the following notes should be considered.

(a) The equation does not show inhibition by excess of tyrosine, since it should be a rational polynomial with at least


1:2 degree in tyrosine. ( b ) The kinetic constant VL,, (Equation 6A) and K;f, (Equa-

tion 7A) have the same denominator, as corresponds to an inhibitor stoichiometric with the substrate.

( c ) The analytical expression of kzt (Equation 6A) is apparent, since kZt depends not only on rate constants but also on equilibrium constants, whereas kEL depends only on rate constants (Equation 14A).

TABLE I1 Values of the kinetic constants of the melanin biosynthesis

pathway determined from experimental data and calculated from simulation data

constants experimental data simulation data Kinetic Value determined from Value calculated from

0.18 f 0.02 0.20 f 0.01 0.13 f 0.01 0.12 f 0.01 1.87 ? 0.20 1.97 f 0.11

(1.28 f 0.06) X lo-' (0.99 f 0.05) X lo-* (1.75 0.10) X 10" (1.50 f 0.04) X 10" 8.03 f 0.10 6.24 f 0.05

107.40 f 1.70 90.20 f 1.60 (5.70 ? 0.04) X lo-' (5.96 k 0.02) X lo-'

(5.30 f 1.50) X lo-' (3.96 f 0.83) X lo-'

"Values referred'for active monomer (3).

0.4 7

0 1 .o 2.0 [Dl, (mM)

[Dl, (mM) FIG. 7. Plot of V& versus [Dl,,. A, experimental assays: 0,

experimental data; 0- - -0, calculated data using initial estimations for the nonlinear regression fitting; U, calculated data using the final estimations from the nonlinear regression fitting. B, simulation assays: 0, simulation data; 0- - -0, calculated data using the initial estimations for the nonlinear regression fitting; ."-., calculated data using the final estimations from the nonlinear regression fitting. [El0 = 1.6 nM.

( d ) Experimental evidence supports KTxy, K& << K1, Kz (8). It has been proposed that k3 << k7 (32,42) in the turnover of the diphenolase activity. This means that the analytical expressions of the kinetic constants can be simplified (Equa- tions SA, SA, 14A, and 15A).

(e) The catalytic efficiencies of tyrosinase on tyrosine and dopa are related through the ratio: ( V L , J K ; f , ) / ( V k x / K g ) = (2/3)R (Equation 18A).

The kinetic analysis of the melanin biosynthesis pathway from tyrosine (Scheme IV) and from dopa (Scheme 11) are based on the rate ratios of Equations 3 and 4, respectively. The computer simulation of both schemes does not use any starting assumption, but directly accomplishes the numerical integration of the corresponding system of differential equations (see "Experimental Procedures"). Thus, the contrast between experimental and simulation assays is of use in verifying the validity of the kinetic analysis and provides a quantitative support to the reability of the reaction mechanisms proposed for the enzymatic steps of the pathway.

Spectrophotometric assays of dopachrome production have led to sets of V6, uersus [ T I o (Fig. 7A) and V& uersus [Dl0 (Fig. SA) values. These data have been fitted by nonlinear regression (56,57) to Equations 5A and 13A, respectively, and the corresponding kinetic constants have been determined (Table 11). From oxymetric assays, the value of KII: has been obtained (Table II), by using the integrated form (58) of its Michaelis Equation 16A). In addition, parallel simulation assays have been carried out (Figs. 7B and 8 B ) yielding kinetic

0.03 1-

0- 0 1.0 2.0

[TI , (mM)

v B

0- 0 1 .o 2.0

[TI, (mM) FIG. 8. Plot of V& versus [m,,. A, experimental assays: 0,

experimental data; 0- - -0, calculated data using initial estimations for the nonlinear regression fitting; M, calculated data using the final estimations from the nonlinear regression fitting. B, simulation assays: 0, simulation data; 0- - -0, calculated data using the initial estimations for the nonlinear regression fitting; ."-., calculated data using the final estimations from the nonlinear regression fitting. [Dl0 = 9 p M and [El0 = 1.6 nM.


constants similar to that calculated from experimental assays (Table 11). Note the verification (Table 11) on the simplified expressions corresponding to Vz,,, KE, V”,.,, Kg and K$ regarding Equations 8A, 9A, 14A, 15A, and 17A, respectively. The expression KE = K , is equivalent to a true inhibition constant, the dissociation constant of the dead-end complex EmetT, whereas Kf; l= K2 is the dissociation constant of a true enzyme-substrate complex EmetD (Scheme IV). In both cases, the overall affinity of tyrosinase toward tyrosine and dopa is determined by E,,,, with lower affinity than Eoxy (8). Fur- thermore, kg, = k3, whereas k:, = (2/3)k3(Kl/K2)R, revealing that k:, is not a true catalytic constant, since it depends on equilibrium constants as well as on the experimental ratio R = 0.057. Note that the value of k z t is lower than that of any rate constants of transformation steps of the reaction mechanism (Table 11).

The value of R (Table 11) is equivalent to the experimentally obtained value (Fig. 3B) according to Equation 5, and also fulfills Equation 18A. This low value of (2/3)R implies the lower efficiency of tyrosinase on tyrosine than on dopa, due to the significant portion of tyrosinase as dead-end complex E,,,T in the monophenolase activity of tyrosinase (Scheme IV) .

In conclusion, the monophenolase activity of tyrosinase can be described by a reaction mechanism (Scheme IV) involving three enzymatic forms with one single active site, two overlapping catalytic cycles, and one dead-end complex. This reaction is coupled to a series of nonenzymatic reactions from o-dopaquinone-H+ which yield dopachrome and regenerated dopa, until the overall steady-state of the pathway is reached. To maintain the steady-state, the enzyme must realize a global turnover involving two turnovers in the hydroxylase cycle for each one in the oxidase cycle. This determines the ratio between the rates of the different steps, which can be used to deduce an analytical expression for the rate in the steady- state of the pathway, leading to its quantitative characterization.

Acknowledgments-JosB Neptuno Rodriguez Lbpez has a fellow- ship from the Comunidad Aut6noma de Castilla-La Mancha. The authors are grateful to Dr. Marino Baiibn for the technical assistance in HPLC determinations.

APPENDIX’

Steady-state Rate for the Monophenolase Activity of Tyrosinase

The melanin biosynthesis pathway from tyrosine starts with monophenolase activity of tyrosinase (Scheme IV). In the steady-state (Equation 3) , it is fulfilled that

V & I = kdEmetD1 + MEoxyDI = 4MEoxyD1, (1.4)

since 2QH yields 1DC (Scheme IV) V& = V&/2. By applying the steady-state approach (58) to the different intermediate species and solving the corresponding system of linear equa-

~~ ~

The notation and definitions are as follows: T, L-tyrosine; D , L- dopa; QH, Q, o-dopaquinone-H’ and o-dopaquinone, respectively; L, leukodopachrome; DC, dopachrome; HD, topa (~-2,4,5-trihydroxy- phenylalanine); PQ, p-topaquinone [5-(2-carboxy-2-aminoethyl)-2- hydroxy-1,4-benzoquinone]; [A, concentration of the species X during the course of the reaction; [XI$., concentration of the species X during the steady-state of the reaction; [Ao, initial concentration of the species X in the assay medium; E, tyrosinase; E,,, oxidized form of tyrosinase with Cui’ in the active site; Ered, reduced form of tyrosinase with Cui in the active site; Emet, mettyrosinase (Eox); Edeoxy ,

deoxytyrosinase (End); E,,,, oxytyrosinase (Eredo2 or E&): V ~ C , V&, steady-state rate of the production of DC from T and D, respectively; V&, V&, steady-state rate of oxygen consumption from T and D , respectively; k, ( i = 1-8), rate constants of the reaction

tions, the analytical expressions for V&! can be derived as follows,

where

L Y ~ = 2kakk7ks(k5 + k-4)KI

P o = ksk-a(k7 + k-d(k.5 + k-4lK1

PI = 3k3k~k7(k5 + k-4)K1 0 2 = ks(k5 + k-,)KI[ks(ki + k-6) + 3kckXzI

Pa = kcks(k3 + 3k,)(kz + k-,)Kj

P, = 3ksk,ks(ks + k-4)KZ + k3k4ks(k7 + k-dK1.

(3.4)

Mushroom tyrosinase is saturated at 0.26 mM O2 (33) and under these conditions (see “Experimental Procedures”), Equation 2A becomes

(4.4)

From experimental and simulated data (Fig. 3B) , it has been obtained the linear ratio: [Dlss = R [ q o (Equation 5), which can be introduced into Equation 4A yielding

The above expressions of these overall kinetic constants can be simplified by taking into account several experimental data as follows. (a) R = [D]../[T10 = 0.057 (Fig. 3B); ( b ) Kg, , Ktxy << K, , K2 (8); (c) k3 << k7, k3 being the limiting step in the reaction mechanism of the diphenolase activity of tyrosinase (32, 42). Therefore, from Equations 6A and 7A, VI,, and KE can be simplified to

V L 2. (2/3)ka(KdK*)R[EIo (8.4)

KK = K , (9.4)

due to (3k7K,K,T., + k3KlKf , )R << (3kjKzKL, + k3KlKfx,) and k3K,Kfx, << 3k7K2Kgx, since k3 << k7 and K I K f x y = K2Kgx,.

mechanism of tyrosinase; K , = k,/k-9, dissociation constant of the deprotonation/protonation equilibrium between QH and Q; klo, cyclization constant of Q into L; kn, rate constant of the production of DC + D from L + QH; k,,,, apparent constant for the transformation of QH into D + DC + H’; K1, K 2 , dissociation constants of Emet toward T and D , respectively ( K l = k- , /k l , K2 = k-z/kZ); KTxy, Kiiy, Michaelis constants of E,,, toward T and D, respectively ( K Z , = (k-4 + ks) /k4 , Kfxy = (k-6 + k , ) / 4 ; Vgax, V:ax, maximal steady-state rates of E toward T and D , respectively (Vz , . = k z t [E]0, V k x = kg, [E],); KK, K i , Michaelis constants of E toward T and D , respectively; VL.,/K;I;, VD,.,/KZ, catalytic efficiencies of E toward T and D, respectively.


Steady-state Rate for the Diphenolase Activity of Tyrosinase REFERENCES

The melanin biosynthesis pathway from dopa starts with diphenolase activity of tyrosinase (Scheme 11). In the steady- state, it is fulfilled that Equation 4

V & = k,[EmetD] + k,[Eox@l = 2h[EmetD] (IOAI

since 2QH yields 1DC (Scheme 11) V& = V&H/2. By applying the steady-state approach (58) to the different intermediate species and solving the corresponding system of linear equations, the analytical expressions for V& can be derived as follows,

where

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8 - ‘ - kdk, + k7)

kdk7 + k-6) + kiKz kdk3 + k7) k3 + k7 8 2 =

Mushroom tyrosinase is saturated at 0.26 mM O2 (33). Under these conditions (see “Experimental Procedures”), Equation 11A becomes the following.

Simplifications of V”,,. and KE-The above expressions of these overall kinetic constants can be simplified by taking into account several experimental data such as K& << Kz (8) and k3 << k7 (32, 42). Therefore, from Equations 12A to 13A, V”,,, and Kfr: can be simplified to the following.

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Therefore, since R = 0.057 (Fig. 4B), tyrosinase shows a lower efficiency on tyrosine than on dopa.

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