Analysis of a Kinetic Model for Melanin Biosynthesis Pathway

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THE O U R N A L OF BIOLOGICALHE MIST R Y

1992 by The American Society

for

Biochemistry and Molecular Biology, Inc

Vol. 261, No.

6 ,

Issue

of February 25, p. 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,nd

Francisco Garcia-Canovaspll

Fr o m the $Departamento de Quimica-Fisica, Escuela Universitaria Politecnica de Albacete, Universidad de Castilla-La Mancha,

Albace te and the §D epartam ento de Bi oquim ica Biologiu Molecular, Facultad de Biologiu, Universidad de Murcia,E-30100

Espinardo, Murc ia , Spain

Thekineticbehavior of the melaninbiosynthesis

pathway from L-tyrosine up to dopachrome has been

studied from experimental and simulation assays. The

reaction mechanism proposed

is

based on

a

single ac-

tive site f tyrosinase . The diphenolase andonophen-

olase activities of tyrosinase involve one single (oxi-

dase) and two overlapped (hydroxylase and oxidase)

catalytic cycles, respectively. The stoichiometryf the

pathway implies that one molecule of tyrosinase must

accomplish two turnovers in the hydroxylaseycle for

each one in the oxidaseycle. Furtherm ore, the steady-

sta te rat es f dopachrome production and

2

onsump-

tion from tyrosine and L-dopa, also fulfill the stoichi-

ometry of the pathway: V /V = 1.5 and V g J V k

=

1.0,where T represen ts L-tyrosine, DC represents do-

pachrome, and

L)

epr esents L-dopa. It has been ascer-

tained by high performance iquid chromatography

that in the steady-state, quan tity of dopa is accumu-

lated

([DlBB)

hich fulfills the constant ra tio

[DlBB

R[mlo.

aking this ratio into account, an analytical

expression has been deduced for the monophenolase

activity of tyrosinase.In hisexpression kTat = (21

3)k3(K1/K2)R,evealing thatkTatis not a true catalytic

constant, since itlso depends on equilibrium constants

and on the experimental

R

=

0.057.

This low value

explains the lower catalyticfficiency 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

s

dead-end complex

EmetT

in the steady-state of th e monophenolase activity of

tyrosinase.

M elan ins are eterogeneous polymers of polyphenolic char-

acterand ittle defined struc ture with color varying rom

yellow t o black 1).Melanins o r ig inate the enzymat icrown-

ing in frui ts and vegetables a s well as the pigm entat io n of

animals . Hum an deficiency in melanins causes albinism and

vit il igo, and grea t in terest has beenhown in the involvem ent

of melan ins in malign ant elanomes, th e carcinogenic tum ors

of the skin. The re have also been s tudie s on he possible

relat ionship between neuromelanins and damage of neurons

and their selective vulnera bility in Parkin son’s disease (2).

Melanogenesis s tarts with the oxid at iony

O 2

of monophenols

and/or o-diphenols th at yield the corresponding o-quinones,

which evolve through coupling nonen zym atic reactions o-

* This paper has been partially supported by a grant from the

Comisi6n Interministerial de Ciencia y Tecnologia (Spa in), project

CICYT ALI89-674. Th e costs of publication of thi s 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 o indicate thi s fact.

Y

T o

whom the correspondence should be addressed.

ward the formationof melanins 1).

Tyro sinase mon ophen ol, L-dopa:oxygen oxidoreductase,

EC 1.14.18.1) is a copper enzyme pr ese nt in microorganisms,

plants , and animals . Different tyrosinases obtainedrom sev-

eral biological sources have simila r stru ctura l and func tiona l

characteris t ics (3). The act ive s i te of tyrosinase consis ts of

two copper a toms and th ree s t a tes , “met ,”deoxy,” an d “oxy”

(4-11). S tructu ral models for the activ e site of these three

forms of tyrosinase have beenroposed (12-14) and con firm ed

by t ransformations into other derivat ives (15-17). T he mon-

ophenolase activity of tyrosinase iscoupled to i ts dipheno lase

act iv i ty and the nonenzymat ic reac t ionsrom the correspon d-

ing o-quinones. T hes e processes ca n be studie d by using a

“bottom-up’’ approa ch, in order o ob tai na successful insight

into the increasingomplexity of th e pathway.

T he o-quinones suffer nonenzymatic breakdown through

polymerizat ion a nd react ion with a number of reagents such

as inorganic ions, reductant agents , hiol and amino com-

pounds, and biological macromolecules (1, 3, 4). The amino

group of the side chain of o-dopaquinone is involved in an

intramolecular 1,4-addi t ion f Micha el into the enzene ring,

causing its cyclization into leukodopachrome’ (4). This inter-

mediate is quickly oxidized to dop ach rom e by an othe r mole-

cule of o-d opaq uinone -H+, which is reduced t o L-dopa

(Scheme I . T hi s process has been kinetically charac terized

from spectrophotometric data

(18)

and has also been verified

from studieswithelectronspin esonance (19) and pulse

radiolysis (20) techniq ues. On the other hand, a similar se-

quence of react ions has been reported for noncycl izable

o -

quinones, s tart ingwith the intermolecu lar addi t ionf nucleo-

philic reagents (21, 22). In act , he hydroxylation of

u-

dopaquinone-H’ can also be significant

at

acid pH (Scheme

I),

as has been detected(23)and kinetically charac terized

(24). Thus, the elanogenic o-quinones evolve through cycli-

zation and hydroxylation branch es involving regeneration of

the respectiveo-diphenol, bu t he hydroxylation bran ch is

only significant at acid pH, as has been detected in melano-

somes an d melanome cells (25).

A

similar kinetic behavior

has been d etected and a nalyzed from a-methy ldopa (26, 27)

an d dopamine

28,

29).

Once the nonen zym atic conversion of o-dopa quinon e-H+

up todopachrome has beenclarified (Scheme

I),

it is possible

to s tudy the diphenolase act ivi tyf tyrosinase. The s t ructural

mec hanism of this reac tion has bee n widely studied (4, 13,

30-32), and the three form sf the enzyme considered Schem e

11). Early kinet ic s tudies into the s teady-statef the pathway

’

The abbreviations and trivial names used are: leukodopachrome,

2,3-dihydro-5,6-dihydroxyindole-2-carboxylate;

yrosine, L-tyrosine;

dopa, ~-3,4-dihydroxyphenyIalanine;-dopaquinone, 4-(2-carboxy-2-

aminoethyl)-1,2-benzoquinone;opachrome, 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 -c

HO -7 -2 0

~~~

T OH

E

Pa HD

SCHEME.

Sequence of rea ct ions

of

the melanin biosynthesis

pathway f rom tyros ine up

o

dopachrome.

k k k

k,

k - 2 k.8 k.. T m et

met

+

D EmstD

Ersc~ 0 2 orv

+

DE

orvD

OH QH

SCHEME1. Reaction mechanism for the diphen olase ctivity

of tyro sinase, coupled

to

nonenzyma tic react ions from o-do-

paquinone-H+ up

to

dopachrome

in

the melanin b iosynthesis

pathway.

2 ~ n

+ D

+n+

SCHEME

11. General eact ion mechanisms for yrosinase

activities

on

tyrosine and dopa.

repor ted an apparen t p ing-pong mechanism

33,34),

a part ic-

ular case of the t rue Ter Bi m echanism Uni Unii Uni Ping

Pong, with two subs trates and two produc ts equal between

them

35).

This rea ct ion me chanism has be en useful for t h e

kineticchara cterization of the nhib ition of tyrosinase by

halides

36)

and by s low binding inhibi tors such as m-cou-

maric acid and mimosine

37, 38).

Fur thermore , th i s reac t ion

E

red

+

0 2

ka

EoxvT

QH

ir:

La,,

D

2 QH +

D

+

H*

DC

SCHEME

V. Reaction mechanism for themonophenolase

ac-

t ivity of tyrosinas e, coupled

to

nonenzymatic react ions from

o-dopaquinone-H+ up to dopachrome in the melanin biosyn-

thesis pathway.

mechanism has served as the ba seor the u nders tand ing and

kinet ic characterizat ion

of

th e suicide inactivation of tyrosin-

ase by o-diphenols such

as

catechol , L-dopa, and dopam ine

39-43).

Th is process is not s ignificant in the t ime rangef a

few minutes , as is usual in s teady-state kinet ic s tudies , but

must be tak en into account to preven t biased recordings of

enzymatic activity.

The above advancesconcern ing heact ivesite of me t,

deoxy, and oxy forms of tyrosinase, the nonenz yma tic reac-

t ions from o-dopaquinone(Scheme I) and he d iphenolase

activity of tyrosinase(Scheme 11) have notbeen properly

considered in any paper on i ts mono phenolase a ct ivi ty

44-

46).

The s t ruc tura l mechanism

for

the m onophenolase ac t iv i ty

of tyrosinase hasbeen widely studied

4,13,30-32)

by consid-

ering the three forms of the enzyme (Scheme 111). Several

kinet ic s tudies

of

the s t eady-s ta te o f the pa thway repor t the

appare nt inhibition by a n excess of tyrosine

31, 44-46)

a s

well as the lower catalytic efficiency of tyrosinase on mono-

phenols than on-diphenols

(8).

Fur thermore , the appearance

of a agperiod has been reported

4, 14, 47-51).

This ag

period depend s on the enzyme and tyrosine concentrat ions,

as has been described from a simplified model

of

Scheme

IV

for frog epidermis tyrosinase

52).

In th i s paper

52)

nei ther

was the oxygen consum ption aken ntoaccount ,nor he

turnover of the enzyme in the pathwa y established. For this

reason no analytica l expression was derived for the mo no -

phenolaseactivity involving the hre esubstrates yrosine,

oxygen, and dopa.

The aim of this paper is the quant i tat ive characterizat ion

of thekin etic behavior of th e monophenolase activity of

tyrosinase. The react ion mechanism (Scheme

V)

involves all

the essen t ial s teps of the c atalyt ic cycle (Schem e 111) and is

coupled to the nonenzymatic react ions fromo-dopaquinone-

H' that yield dopachrome and regenerated L-dopa. The reli-

abi li ty of this react ion m echanism and the establ ishmen t of

the enzymat ic tu rnover in the pa thway has beenverified by

experimentalandsimulation assays. In hi s way, a valid

analytica l expression

for

the s teady-state rate

of

the mono-

phenolase activity

of

tyrosinase

is

derived for the fi rs t t ime

in he i terature.T h ecorrespondingkinet icconstants are



Kinetic

M e c h a n i s m

of

T y r o s i n a s e

dete rm ined and their physical significance discussed. In ad-

di t ion,as egards he elat ionship between theanalyt ical

expressions obtained or the m onoph enolase and d iphenolase

activities, the lower catalytic efficiency of the enzyme on

monophenols than on d iphenols i sxplained.

EXPERIMENTALPROCEDURES

Materials-Mushroom tyrosinase (3300 units/mg), tyrosine, and

dopa were purchased fromSigma. All other chemicalswere of analyt-

ical grade and suppliedby Merck. Mushroom tyrosinase was purified

by the procedure of Duckworth and Coleman (33). Protein concen-

trat ion was determined by a modified owry method (53). Th e enzyme

concentration was calculated takinga value of

M ,

120,000.

Kinetic Assays-The dopachrome accumulation was spectropho-

tometrically followed at 475 nm c = 3600 M-' cm ) using a Perkin-

Elmer Lambda-2 spectrophotometer interfaced on-line with an AM-

STRAD PC2086 computer. Th e reaction medium was 10 mM sodium

phosphate buffer, pH 7.0. Toestimate hekineticparameterson

tyrosine, care was taken hat recordings reached steady-s tate cor-

rectly, with no significant consumption of substrate, suicide inacti-

vation, or dopachrome breakdown. This aspect was solved by the

addition to the reaction medium of a [Dl0

<

[D],,/[qo, as well as

shortassays imes. neachassay he act that he pathway had

reached the steady-sta te was checked by quantifying the [Dl accu-

mulated in the reaction medium. Th is was done by measur ing the

absorbance increase a t 475 nm produced at 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 n a Beckman

System Gold liquid chromatography system equipped with a pump

model llOB for socratic 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 introducedvia a fixed-

volume injector (20 pl) Rheodyne. The compounds were sepa rated in

an Ultrasphere-ODS 4.6 X 250 mm; i.d. 5 pm) reversed-phasecolumn

and eluted at flow rate of 1 ml/min with ammonium acetate 50 mM

and Na2-EDTA, 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.Th e 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 Na I0 4 in a 1:2 stoichiometry (dopa/

NaI04).

Oxygen Determination-Oxygen consumption was followed by a

Hansatech DWoxymeter, based on the Clarklectrode. Temperature

was controlled at 25 C using a Haake D1G circulating bath with a

heater/cooler and checked using a Cole-Parmer digital thermomet er

with a precision of kO.1 C.

Simulated Assays-The kinetic behavior of the reaction mecha-

nism is described by a system of differential equations, whose nu-

merical integration was carried out by using the predictor-corrector

algorithm of Adams-Moulton, starting with a fourth order Runge-

Kutta method (54). Th e algorithm was implemented andcompiled in

TurboBASIC 1.0 on a n I NVES PC-640A computer (IBM AT-com-

patible) with an Int el 80287 arithmetic coprocessor.

The reaction mechanism of the monophenolase activityf tyrosin-

ase (Scheme IV) involves the di fferentia l equations as ollows.

[&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 ~ ) [ E o x y T I

4QHl = kdEmetD1+ k7[Eox,Dl

-

kapp[QHl

P C 1

= 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 accordancewith

experimental data.

Th e 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 . Th e [Dl was considered constant

throughout the si mulations, in ccordance with experimental data.

Assignment

of

Constants-The values of the equilibrium and rate

constants of the model were assigned by taking nto account he

kinetic analysis of the mechanism s as well as he experimen tally

determined kinetic parameters. By nonlinear regression of the Vo

values versus [Dl, and

[ g o ,

the parameters

K g ,

VgaX,

Z ,

and

V z e x

were determined. The fitt ing f the integratedMichaelis equation for

oxygen consumption gave

K?.

From VE., the catalytic constant k3

was determined. For theMichaelis constants of

Eoxy

n tyrosine and

dopa he magnitude order was taken from he iterature (8). kapp

corresponds to the processes of protonation-deprotonation of o-do-

paquinone-H+, cyclization, andheurther xidation-reduction,

which yield the formation of dopachrome and the regenera tion of

dopa in themedium (18). Thus, the setf values for he rate constants

of the reaction mechanisms f tyrosinase was obtained (Table I).

RESULTSANDDISCUSSION

Stoichiometry

of

the Pathway-The mela nin biosynth esis

pathway from tyrosine to dopach rome consis t of enzymatic

react ions of tyrosina se on tyrosine and on dopa yielding

o-

dopaquinone-H', which evolves nonenzy matically tow ard do-

pachro me (Schem e I) . T he verall s toichiometry of the pa th-

way involves, therefore, the catalyt ic turnover of the enzy-

mat ic reac t ions aswell as further nonenzymatic s teps. Thus,

the conversion of tyrosine up to dopach rome isefined by the

following mass balance (Scheme IV);

7'+ E,,,

+

2H+ D + E,,, + H20

D

+

Erne,

QH +

Edeory

+ 2H'

0 2 + Edeoxy E,,,

T + E,,, + 2H' D E,,, H20

D

+ E,,,

QH + Edeory

+

2H'

0 2 +

Ed eo x y

E,,,

2 Q H * D + D C + H +

D

+

Eo,,

+

4H' QH

E,.t

+

2H' 2H20

D

+

E m e t -+QH + Ed eo ry + 2H'

0 2

+

&eoxy E,,,

2 Q H + D + D C + H +

27'

30, +

2DC

+

2H+ 4H20

whereas the mass balance for the conversion of d opa up to

dopachrome (Sche me 11) is as follows.

D

+

Erne, QH + Edeory 2H+

0 2

+

Edeoxy E,,,

D

+

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

2QH D + DC + H'

D

+

0-8

4 U C

+

H'

+

2H20



3804 Kineticechan ism of Tyrosinase

TABLE

Values

of

the rate constants used or simulation

of

the melanin

biosynthesis pathway, taking into consideration the reaction

mechanism

of

tyrosinase

Michaelis

constants

Transformation

constantsonstants

K I = 1.9

X

lo-' M k l = 5.5

x

l o7

M-'

s-' kZi= lo's-'

k-l = 1.0 X 104

s-1

k s = 10

s-'

KE

=

1.4

X

M

k,

=

7.3

X

l o7

M-I

sC1

ki

=

l o 3

-'

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 k s =

5 .5 x

10'

M - I s-1

k-6

=

1.0

X 103

8-1

k-s = 1.0 X 103

s-l

Th e melanogenesis pathway from tyrosine s tarts with the

monophenolase activity of tyrosinase Sche me V), which

consist of two catalytic cycles overlapping through three com -

mon interm ediates . The s toichiome try of the pathw ay (bal-

ance

l )

implies tha t one molecule of tyrosinase m ust accom-

plish two turnovers in the hydroxylase cycle for each one in

the oxidase cycle (Schem e IV). Thus, yrosinaseopera tes

three t imes, twice a nd once thro ugh the s teps control led by

k3, k g ,

an d

k7,

respectively. Th erefore , in the steady -state of

the pathwa y, the ol lowing rate rat ios mustbe fulfilled.

k,[Eme,Dl = (3/2)kdEoxsr l = [&&I ( 3 )

T he reliability of these ratios has bee n erified from simu -

lation assays (Fig.

1, A-C)

in the final s teady-state of the

pathway (from

600 s).

At

a

shorter t ime range

(2.0

s) there is

an early s teady-state restricted to theydroxylase cycle (Fig.

1A)

with

k3[E,,tD]/k5[E,,,~1.0,

which evolves toward the

overal l s t eady-s ta te of the pa thway with k3 [E , , t~ ] /k 5[ &, , ,~

=

1.5.

On the other hand, the melanogenesis pathway from

dopa tarts with the iphenolase ct ivi ty of tyrosinase

(Scheme 11),defined only by the oxidase cycle and on esingle

turnover balance 2) . In hestead y-state of thepathway,

therefore, the rate rat ios verified as follows,

k,[EmetDl =

k 7 [ E o x y D ] ,

(4)

in accordancewith simulationassays (Fig.

1 D ) .

T h e a t e

rat ios (Equat ions

3

a n d

4)

are useful in the derivat ion of th e

steady-state rate equat ions for the catalyt ic act ivi t ies of ty-

rosinase (see "Appendix").

The s toichiome try of the melan in biosynthesis pathw ay

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 simulat ion (Fig.

2B)

assays. Thes e results Figs.

1

an d

2)

sup po rt the eliability

of the react ion mech anisms roposed for the monophenolase

(Scheme IV) and d iphenolase (Scheme

11)

activities of tyro -

sinase, involved in the pathw ay u nder s tudy (Schem e I) .

Accumulation of [Dl,-The ope ratio n of th e melanog enesis

pathway from tyrosine (Scheme IV) can be monitored

at 475

nm, and showsa t ransie nt pha se hat evolves toward he

overal l s teady-state of the pathw ay,with linear produ ction of

dopachrome (Fig.

3A).

Th is process can also be followed in a

discont inuous way by t i t rat ion of the [Dl accu mulated in the

assay medium w ith Na I04. Thu s, a s igmoid pat tern of

[Dl

uersus t ime, whose final plateau corresponds to the level

of

[Dl,, is obtaine d (Fig.

3A) ,

s imul taneous ly wi th he inear

formation of dopachrome. Th e [Dl ,, i s not depen dent on E],

(Fig.

3 B ) ,

whereas i t is proport ional to

[ T I o

(Fig.

3 B ) ,

[Dlss

=

R [ r l s S R[ , ( 5 )

since the consum ption of tyrosine is negligible during he

assay time (Fig.

3A).

The same behavior and dependencies

were obtained from simulat ion assays (resul ts not shown).

The t ransient phase of the pathw ay (Fig.

3A)

involves the

regenerat ion of dopa in the nonenzy matic react ions from

o-

dopaqu inone-H +, as well

as

the reported competi t ion of ty-

rosine and dopa on the

Emet

n d

E,,,

forms

14)

(Scheme IV),

which is consis ten t w ith the nondep enden ce of

[Dlss

on

[El0

(Fig.

3B) .

Thus , the increas ingevels of [Dl remove

Emet

rom

~

0 600 1200

t

(SI

0

600

1200

t s )

0 50 100

t

(SI

FI G . 1. Evolution with time

of

severa l rate ratios of the

catalyt ic

cycle. A-C, monophenolase activity (Scheme IV), with 0.2

mM tyrosine, 0.26 mM

O,,

and 0.1 P M tyrosinase. D, iphenolase

activity (Scheme

II),

with

0.2

mM dopa, 0.26 mM

0 2 ,

and

0.1

y M

tyrosinase.



Kineticechanism

of

Tyrosinase 3805

2

V

>

\

P

2

>

\

9

1

6

O) CD l , mM)

FIG. 2.

A ,

experimental results of relation between steady-state of

2 consumption and dopachrome formation:

0 ,

dependence on [Dlo ,

with [E10 = 1.6 nM; A, dependence on [qo ith [ E ] ,=

5.0

nM; B ,

relation between steady-state rates

of

0,

onsumption and dopa-

chrome formation obtained by simulation: 0 , dependence on [Dl0 ,

with [ E ] = 0.1

PM;

A, dependence on [qo,ith

[E l o

= 0.1 FM.

the dead-en d omplex

EmetT,

ielding

E,,,&,

which is involved

in the xidase cycle (Scheme

IV).

This increases the operat ive

[E] s wel l as the form ation of o-dopaquinone-H+ and do-

pach rom e up to the at tainm ent f the overal l s teady-state of

the pathw ay Fig. 3 A ) .Therefore , themon'ophenolase activity

of tyrosinase (Scheme IV) s tarts with the ingle operat ion of

the hydroxylase cycle an d evolves toward the s teady-state,

with two tur nov ers in the hydroxylase cycle for each one in

the oxidase cycle, as wel l as a ne t decrease in the early EmetT]

due to the regenera t ionf dopa in the nonenzymat ic reac t ions

from o-dopaquinone-H+. For this reason, t higher values of

[7 l0

there i s an n crease n he ear ly

[EmetTJ,

nd grea ter

values of [Dlssare required for the at ta inm ent of the s tea dy-

sta te of the pathw ay Fig.

3 3 ) .

Th e removal of

Emet

rom the dead-endomplex EmetTould

be c arried o ut by any dipheno l presen t in the assay medium

such as d opa or eukodopachrome, according to the ir respec-

t ive levels in the s teady -state of the pathw ay (Schem e IV).

The quan t i ty o f [Dl,,as been experimental ly determined

(Fig. 3, A a n d B ) , whereas leukodopachrome has not been

detected in the react ion medium 18, 4). T he value of [L],,

can be calcu lated from the ol lowing.

[LI = k1o[Q1

-

~ I I [ Q W [ L I0

6 )

Therefore

(24),

I I

0.11

I

0

1.0 2 .o

0 )

TI, mM)

FIG.

3.

A ,

experimental results of time course of the accumulation

ofdopachrome

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,, ith ( E ] ,= 5.0 nM; A , dependence on

[E l o

with [ T I o = 1 mM.

since k-9[H']

s ,,

a t p H

7.0,

and where

k,= 0.41/s (18)

an d

k > lo9 M

s-'

(55).

Thu s, the con tributio n of leukodopa-

chrome

(14)

perhaps could be s ignificant early n the react ion,

but isnegligible

at

the s t eady-s ta te f the pa thway , d ue to the

low value of

[L],,

Equat ion

7).

Induct ion Periods in the Product ion

of

Dopachrome

f r o m

Tyrosine-The induction period observed in the pro duc tion

of dopachrome rom tyrosine (Fig. 3 A ) i s a ag t ime hat

increaseswhen

[El0

is decreased or

[ T I 0

is increased

(52).

Several tentative explan ations for these prope rties have been

proposed by different authors . Thus, a m odel h as been pro-

posed

(48)

with two consecutive enzym atic reaction s in dif-

ferent catalyt ic s i tes and which predicts an exponent ial ac-

cum ulation of [Dluersus t instead of the exp erim enta l igmoid

pat tern (Fig. 3 A ) . A similar model

(33)

proposes th e direc t

conversion of tyrosine into dopa and the further oxidat ionf

dopa, with no net accumulat ion f dopa in the medium. Other

authors have suggested the slow generation of dopa from

tyrosine through no nenzymatic react ions or the s t rong bind-

ing of dopa to tyrosinase 50), s well as the act ion f dop a as

positive effector on one allosteric site of tyrosinase

(51),

with

no support ing evidence from structural or kinet ic s tudies in

ei ther case. Fur therm ore the lag period (Fig.

3 A )

has been

at t r ibu ted to the conversion of

E oxy

nto

E,,,

(14),

but th i s

process is fas t and occurs in the milliseconds range

(8).

All the interac t ions etween tyrosinase, tyrosine, and dopa

in the monophenolase act ivi ty of tyrosinase take place a t th e

binuclear coppersite of tyrosinase , withou t llosteric phen om-

ena

(14).

The lag period, therefore, involves (Scheme

IV)

t h e

regeneration of dop a in the nonenz ymatic react ions from

o-

dopaquinone-H', as well as the emoval of

E,,,

f rom the dead-

end complex

E,,,T

to incorporate

E,,,D

into the xidase cycle

(52) .

T he above interpretat ion suggests th at th e ag periodof the

monophenolase activity of tyrosinase on tyrosine should be

shortened by the addi t ionof dopa a t the s t ar tf the react ion,

due to he lower t ime required or the at tainm ent of the

corresponding

[D],,.

n fact , there are three possible ini t ial

conditions:

[Dl0< [Dl,,

a n d

[Dl0> [ Dl s s

ead to the l ag and



3806

Kineticechanism of Tyrosinase

burst nduction period, respectively, wh erea s the con dition

[Dl0

=

[Dls3does not originate induct ion period. These three

caseshave beenobta ined nexperimental (Fig.

4)

a n d n

simulation (Fig.

5 )

assays. The

parallel

straig ht lines show

that the prod uct ion of dopachrome evolves towa rd the same

steady-state of the pathwa y (Figs.

4

an d 5), only d etermined

by[E]o and

[q0.

PLC has beenapplied toexper imenta l

assays (Fig. 4)with the three ini t ial condi t ionsFig.

6,

A-F),

ob ta in ing the samealue of

R

(0.057),

which is also equiva lent

to that calculated from simulat ion assays (Fig. 5 ) . Fur ther-

more, the rate rat ios (Equat ion

3)

are only accomplished in

the s t eady-s ta te o f the pa thw ay. Note tha t whe n no d opa i s

added to themedium the consum ption f tyrosine may become

significant, making the mea surem ents for kinetic studies dif-

ficult (Fig. 6,A a n d B ) . However, when the reac tion

starts

with

[q0

nd [Dl0,

[ l ‘ ls8 [q0

Fig.

6, C -F )

and , herefore ,

thi s procedure is recommendable for kinet ic s tudies on the

pathway (Scheme IV). The [D l0 chosen c an be n ear to [Dlss

determined by t i t rat ion with NaI04

or

by HPLC.

There fore, an nappropriate experim ental design (44-46)

has led to misleading conclusions about the monop henolase

act ivi ty of tyrosinase. There are several points which arise

from these s tudies andwhich shou ld be comm ented on:

a)

The appearance and disappea rance of a lag period

at

different p H values may be due to th e se of a slow discontin-

uous method for themeasu reme nt of themonophenolase

activity. Thi s procedure is not sui table for the determ inat ion

of t ransient pha se para meters , su chs the lag period.

( b )

The observed inhibition of tyrosinase by an excess of

tyrosine is possibly due to the se of the sam e assay t ime

or

different

[TI,.

hus,

at

high

[T I o ,

the lag eriod increases and

“appare nt s lopes” can be determined, which a re lower than

the t rue s t eady-s ta te ra tes .

(c) Th e use of high conc entrat ion of ascorbic acid causes

the reduct ion of o-dopa quinone-H + to dopa,hose continuous

accum ulat ion preven ts the at tainment of s teady-sta te in th e

pathway (Scheme

I).

Fur thermore , th i s reagen t can or ig inate

“0

lo

t (min)

20

FIG.4.

Experimental results of time course of dopachrome

accumulation, with several values of [Dl0, pM):

a, ;

b, 6; c,

11;

d ,

17;

,

22.

In all ca ses 0.2 mM tyro sine , 0.26 mM

02

n d

5.0

nM tyrosinase was used.

0

t (m i d

15

FIG.5.

Simulation results of time course

of

dopachrome

accumulation, with several values of [Dl0

(pM): a ; b, 11; ,

22.

In all cases

0.2

mM tyrosine, 0.26 mM

02

n d

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 ~ ) :

-B,

0;

C-D,

11; and

E-F, 22. Re age nts: 0.2 mM tyro sine , 0.26 mM

02

nd 5.0

nM tyrosinase. 1, tyrosine; 2, dopa; an d 3, dopachrome.

th e reduction of Emetnto Edeoxy, hich m odifies the enzymatic

turnover.

d )

Th e proposal of a model for th e monophenolase activity

of tyrosinase, with pH-de pende nt in terconv ertible forms and

one allosteric site for the inhib ition y an excess of tyrosinase

does not take into acc oun t the ccurrence of Emet,Edeoxy,nd

Enryorms and s no t suppor tedby any struc tural evidence.

Kinetic Analysis-The exp erim enta l and sim ulatio n ssays

carried ou t in the bove sect ions support theeliability of the

proposed react ion mechanism (Scheme IV) an d perm it the

turnover of the enzyme in the pathw ay to be establ ished. The

constancy of

R

is confirm ed and i ts value for this enzyme

calculated (Table 11).Therefore, i tbecomes possible to derive

the corresponding rate equat ion for the s tead y-state of the

pathway, which enables i ts quant i tat ive charac terizat ion.

The k inet ic analysis of the mo nophen olase act ivi ty of ty-

rosinase has not been properly accomplished, since conv en-

tional reaction mechanism s with only one single cycle have

beenproposed, such as bisubstrate ping-po ng and ordered

mechanisms (49). urthermore, kinet ic analysisof the pa th-

way from tyrosineup odopachrome has been at tempted

without taking into considerat ion the regenerat ionf dopa in

the n onenzy matic react ions rom o-dopaquinone-HC(14).

In the “Appendix” are detai led the kinet ic analysesof the

monophenolase (Scheme IV) and of the diphenolase Scheme

11)

activities of tyrosinase, taking into account the turnover

of the enzyme in this pathw ay and the contribut ion of the

nonenzymatic react ions from o-d opaqu inone-H + to the me-

lanogenes is pa thway . The s teady-s ta te ra te V& (Equa t ion

2A) is de pendent on

[Elo

nd on a l l the subs t ra tesnvolves in

the reaction

[ T I o , [Dlsb,

n d

[OZlO

Scheme IV). Moreover, a t

saturat ing condi t ions of [O2 lO and ccording to Equat ion

5 ,

Equation 2A is simplified toEquat ion 5A. However, the

following notes should be onsidered.

a )

The equa t ion does not show inhibi t ion by excess

of

tyrosine, since it shou ld be rational polynomial with at least



Kinet icechani sm

of

Tyrosinase

3807

1:2

degree in tyrosine.

( b )

The k inet i c cons tan t L,, (Equat ion

6A)

a n d K ; f , Equa-

t ion

7A)

have the same denominator, as corresponds to an

inhibi tor s toichiometric with the substrate.

c ) Th e analyt ical expression of

k z t

(Equat ion

6A)

is ap-

parent , s ince

kZt

depend s not only on rate constants but lso

on equi librium constants , whe reas kELdepends only on rate

cons tan t s (Equat ion 14A).

TABLE

1

Values of the k inet ic constants f the melanin biosynthesis

pathway determined from experimental data andalculated

f rom s im u la t i on da ta

constantsxperimentalataimulationata

Kinetic Value determined from Value calculated from

0.18 0.02

0.20 0.01

0.13 0.01

0.12 0.01

1.87? 0.20

1.97 0.11

(1.28 0.06) X lo-

(0.99

0.05)

X lo-*

(1.75 0.10) X

10

(1.50 0.04) X

10

8.03 0.10

6.24

0.05

107.40 1.70

90.20 1.60

(5.70

?

0.04)

X

lo-

(5.96

k 0.02)

X

lo-

(5.30 1.50)

X lo-

(3.96 0.83)

X lo-

"Values referred 'for active m onomer

(3).

0.4

0 1o 2.0

[Dl, mM)

[Dl, m M )

FI G . 7. Plot

of

V versus [Dl,,. A , experimental assays: 0 ,

experimental data; 0- -0, alculated data using init ial estimations

for the nonlinea r regression fitting;U alculated data using

the final estim ations from the nonlinea r regression fi t t ing. B, s imu-

lation assays: 0 , imulation data; 0 - -0, alculated data using the

initial estim ations for the nonlinea r regression fitting; . - cal-

culated datausing the final estimations from the nonlinear egression

fitting. [El 0= 1.6 nM.

d ) Experimental evidence supports KTxy,

K & <<

K1, K z

(8).

t has been roposed that k3

<< k7 ( 3 2 ,4 2 )

in the turnover

of the diphenolase activity. This mean s that the analy t ical

expressions of the k inet i c cons tan t s can beimplified (Equa-

t ions

SA, SA, 14A,

an d

15A).

(e ) T he catalyt ic 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

(Equat ion

18A).

The kinet ic analysis

o f

the melan in b iosynthes i s pa thway

from tyrosine (Scheme IV) and from dopa (Scheme 11) are

based on the rate rat ios of Equat ions

3

a n d 4, espectively.

The com puter s imulat ion of both schemes does not use an y

start ing assumption, butdirectly accomplishes the num erica l

integrat ion of the correspo nding system f different ial equa-

t ions (see "Experime ntal Procedures"). Thu s, he contr ast

between experimentaland imulat ionassays is of use in

verifying th e validity of the kine tic analysis and provides

a

quant i tat ive support to the reabil ity of the react ion mecha-

nisms proposed for the enzym atic s teps

f

the pathway.

Spectrophotometric assays f dopachrome product ion have

led

to se t s of

V 6 , uersus [ T I o

(Fig.

7A)

a n d

V& uersus

[Dl0

(Fig. S A ) values. These data have been fitted by nonlinear

regression

(56 ,57)

to Equat ions

A

a n d

13A,

respectively, an d

th e corresponding kinet ic constants havebeen 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). Inaddition,paral lel imulat ion

assays have been carried out Figs.

7 B

a n d 8 B )yielding kine tic

0.03 1

0

0

1.0

2.0

[ T I , mM)

v

B

0

0 1.o

2.0

[ T I , mM)

FIG.

8.

Plot

of

V versus [m .

,

experimental assays: 0 ,

experimental data; 0 - -0, alculated data using init ial estimations

for the nonlin ear regression fitting;

M

alculated data using

th e final estimations from the nonlinea r regression fi t t ing. B , simu-

lation assays:

0 ,

simulation data;

0- -0,

alculated data using the

initial estimations for the nonlinear regression fitting;

.

cal-

culated datausing the final estimations from the nonlinearegression

fitting.

[Dl0

=

9

p M an d [El0= 1.6 nM.



3808 Kineticechanism of Tyrosinase

cons tan t s s imi lar to tha t ca lcu la ted f rom exper imenta l assays

(Table 11).No te the verification (Ta ble

11)

on the simplified

expressions corresponding to

Vz,,, KE,

V”,.,,

K g

a n d

K

regarding Equat ions 8A, 9A, 14A, 15A, a n d 17A, respectively.

T he expression KE = K , is equivalent to a t rue inhibi t ion

cons tant , the dissociat ion con stant of the dead-en d complex

EmetT,

hereas

K f ; l = K 2

s thedissociation con stan t of a true

enzyme-substrate complex EmetD Scheme IV). In both cases,

th e overall affini ty of tyrosinase towa rd tyrosine and dopa is

determined by

E,,,,

with lower affini ty than

Eoxy

8) . Fur-

thermore, k g , = k3,whereas k:, = ( 2 / 3 ) k 3 ( K l / K 2 ) R ,evealing

t h a t k:, is not a t rue catalyt ic constant , s ince i t depends on

equilibrium constants

as

well as on the experimental rat io

R

=

0.057.

Note that the alue of k z t is lower tha n tha t of any

rate constants of t ransformation s teps of the react ion mech -

an i sm (Table

11).

T he value of R (Table 11)i s equ ivalen t o the xperimental ly

obtained value (Fig.

3 B )

according to Equat ion 5, and also

fulfills Equation 18A. This low value of

( 2 / 3 ) R

mplies the

lower efficiency of tyros inase on ty rosin e th an o n dopa, due

to the s ignificant port ionof tyrosinase as dead-endcomplex

E,,,T

in the mon opheno lase act ivi ty of tyrosinase (Scheme

IV)

.

In conclusion, the mono phenolase act ivi tyf tyrosinase can

be described by a reaction mechanism (Scheme V) involving

three e nzym atic form s w ith one s ingle act ive s i te, two over-

lappingcatalytic cycles, andonedead-en d complex. Th is

reaction is coupled to a series of none nzym atic reactions rom

o-dopaquinone-H+ which yield dopachrome and regenerated

dopa, unt i l the veral l s teady-state of the pathwa y iseached.

To maintain the s teady-state, thenzyme mu st realize aglobal

turnove r involving two turn ove rs in the ydroxylase cycle for

eachone in he oxidase cycle. Th isdetermines he a t io

between the ratesof the different s teps,which can beused to

deduce an analyt ical expression for the rate in the s teady-

state of the pathwa y, eading to i ts quant i tat ive characteriza -

tion.

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.

APPENDI X’

Stea dy-s tate Rate for the Monophenolase

Ac tivi ty of Tyrosin ase

Themelan inbiosynthesispathwa y from tyrosinestarts

with monophenolase act ivi ty of tyrosinase (Scheme IV). In

the s t eady-s ta te (Equat ion

) ,

t

is

fulfilled th at

V & I kdEmetD1 + MEoxyDI = 4MEoxyD1,

1.4)

since

2QH

yields 1DC (Scheme IV)

V& = V&/2.

By applying

the s teady-state approach

(58)

to the d i f feren t in termedia te

species and solving the corres ponding system of linear eq ua-

~~ ~

The notation and definitions

are

as follows: T, -tyrosine;D , L-

dopa;

Q H , Q , o-dopaquinone-H’

and o-dopaquinone,

respectively;

L ,

leukodopachrome; DC, dopachrome; H D , topa

(~-2,4,5-trihydroxy-

phenylalanine); P Q , p-topaquinone [5-(2-carboxy-2-aminoethyl)-2-

hydroxy-1,4-benzoquinone]; A oncentration of the

species

X dur-

ing the course of the reaction; [XI ., concentration of the

species X

during the steady-stateof the

reaction; [Ao

nitial

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

( E o x ) ;

de oxy ,

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&,

teady-state rate

of oxygen

consumption

from

T nd

D ,

respectively;

k ,

i = 1-8),

ate

constants

of the reaction

t ions, the analyt ical 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 tyrosin ase is satura ted at 0.26 mM

O 2 ( 3 3 )

a n d

underhese ondi t ions (see “ExperimentalProcedures”),

Equat ion 2A becomes

4.4)

From e xperime ntal and s imulated data (Fig.

3 B ) ,

i t has

been obta ined the linea r ratio: [Dlss= R [ q o (Equat ion 5 ) ,

which can be introduced into Equat ion

A

yielding

Th e above expressions of these overal l kinet ic constants

can be simplified by taking into acc oun t everal exper imental

dat a as follows. a)R = [D] . . / [T10= 0.057 (Fig. 3 B ) ; b )K g , ,

K t x y<<

K , ,

K 2

8);

(c)

k3

<<

k7, k3

being the l imit ing s tep in

the reac t ion mechanismf the diphenolase act ivi tyf tyrosin-

ase (32 , 42) . Therefore, from Eq uat ions 6A an d 7A, VI,, a n d

KE

can be simplified to

V L ( 2/3 )k a( Kd K* )R [E Io

(8.4)

KK

= K ,

(9.4)

due to

(3k7K,K,T.,

+

k 3 K l K f , ) R<< ( 3 k j K z K L ,+ k 3 K l K fx , )

n d

k 3 K , K f x ,< 3k7K2Kgx, ince k3 << k7 an d K I K f x y K 2 K g x , .

mechanism of

tyrosinase;

K ,

=

k , / k - 9 ,

dissociation constant

of the

deprotonation/protonation equilibrium

between

QH and

Q;

klo, cycli-

zation constant

of

Q into L ; kn , rate

constant of the

production

of

DC D from

L +

QH; ,,,,

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,Ki iy ,

Michaelis constants of

E,,, toward T

nd

D ,

respectively

( K Z , = (k-4

+

k s ) / k 4 , K f x y ( k - 6

+

k , ) / 4 ; Vgax,V:ax,

maximal

steady-state rates

of

E

toward

T

nd

D ,

respectively

( V z , .

=

k z t [E]0,V k x= k g , [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 ,

re-

spectively.



Kinet icechani sm of Tyrosinase 3809

Steady-stateateorheiphenolasectivity of Tyro sinase REF ERENCES

The melanin biosynthesis pathway from dopa starts with

diphenolase activity of tyrosinase (Scheme 11). In thesteady-

state, i t 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 equa-

tions, the analytical expressions for

V&

can be derived as

follows,

where

=

k3

+

4 k 7

8 -

‘

-

kdk,

+

k7)

kdk7

+

k-6) kiKz

kdk3 + k7) k3

+

k7

8 2 =

Mushroom tyrosinase is saturated at

0.26

mM

O 2 ( 3 3 ) .

Under

these conditions (see “Experimental Procedures”), Equation

1 1 A 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 ( 3 2 ,42). Therefore, from Equations 1 2 A to 13A,

V”,,,

and

Kfr:

can be simplified to the ollowing.

V L (k [EIo)/(k3 + k7) kdE10 ( 1 4 4

and

Oxygen Consumption-The

affinity of tyrosinase toward O2

is inversely related with its corresponding Michaelis constant,

which expression can be derived from Equation

1 1 A

at satu-

rating [ D l ovalues as follows,

where

Catalytic Efficiencies of Tyr osin ase on Ty rosin e a nd opa

The catalytic efficiency of tyrosinase on tyrosine

(VLJ

KZ) and on dopa (VD,.JKD,), defined from Equations 8 A to

9 A and 1 4 A to 15A, respectively, are related through the

following expression:

Therefore, since

R

=

0.057

(Fig.

4B),

yrosinase shows a lower

efficiency on tyrosine than on dopa.

1. Prota, G. (1988) Med. Res. Rev. 8, 25-556

2. Langston , J . W. (1988) Trends Pharmacol. Sci .

9,

347-348

3. Robb, D.

A.

(1984) in Copper Proteinsand Copper Enzym es

4. Mason, H. S. (1956) Nature 1 7 7 , 7 9- 81

5.

Jolley, R. L., Jr., Evan s, L. H., and Mason , H.S.

(1972) Biochem.

6. Schoot-Uiterkamp, A. J. M., and Mason,H. S. (1973) Proc. Nat l .

7. Jolley, R. L., Jr. , Evans, L. H., Makino, N., an d Mas on, H. S.

(1974) J . Biol. Chem . 2 4 9 , 3 3 5- 34 5

8. Makino, N., and M ason, H. . (1973)

J.

Biol. Chem.

248,

5731-

5735

9 . Makino, N., McMahill, P., Mason,

H. S.,

and Moss,

T. H.

(1974)

J . Biol. Chem. 2 4 9 , 6 0 6 2 - 6 0 6 6

10. Schoot Uiterkamp, A . J. M., Evans, L.

H.,

Jolley, R. L., and

Mason, H. S. (1976) Biochim. Biophys. Acta 4 5 3 , 2 0 0 - 2 0 4

11. Lerch, K. (1981) in Metal

I ons

in Biological Sys tems (Sigel, H.,

ed) pp. 143-186, Marcel Dekker, Inc., New York

12. Himmelwright, R. S., Eickm an, N. C., Lu Bien, C.

D.,

Lerch, K.

and Solomon, E. I. (1980) J . A m . Chem. SOC. 02, 7339-7344

13. Solomon, E. I. (1981) in Copper Proteins (Spiro, T . G., ed) Vol.

111,pp. 41-108, Wiley-Interscience, New York

14. Wilcox, D. E., Porras, A. G., Hwang, Y. T., Lerch, K., Winkler,

M. E., and Solomon, E. I. (1985) J. A m. C hem.

SOC.

0 7 , 4 0 1 5 -

4027

15. Beltramini, M., Salvato, B., Santa maria , M., and Lerch, K . (1990)

Biochim. Biophys. Acta 1 0 4 0 , 3 65 -3 72

16.

Menon, S.,Fleck, R. W., Yong, G., and Strothka mp,

K.

G.

(1990)

Arch. Biochem. Biophys. 2 8 0 , 2 7 - 3 2

17. Yong, G., Leone, C., and Strothkamp, K. G. (1990) Biochemistry

2 9 , 9 6 8 4 - 9 6 9 0

18. Garcia-Carmona,

F.,

Garcia-Canovas, F., Iborra, J. L., and Loz-

ano,

J.

A. (1982) Biochim. Biophys. Acta

717,

124-131

19. Kalyanaraman,

B.,

Felix, C. C., and Sealey,

R.

C. (1982) Photo-

chem . Photobiol. 3 6 , 5 - 12

20. Land, E. J. , Thompson,A., Truscott , T. G., Subbarao, K. V., and

Chedekel, M. R. (1986) Photochem . Photobiol. 4 4 , 6 9 7 - 7 0 2

21. Cabanes, J. , Garcia-Canovas, F., and G arcia-Carmo na, F. (1987)

Biochim. Biophys. Acta 9 1 4 , 1 9 0- 19 7

22. Garcia-Carm ona, F., Cabanes,

J.,

and Garcia-Canovas, F. (1987)

Biochim. Biophys. Acta

914,

198-204

23. Garcia-Cinova s, F., Garcia-C armona, F., Sanchez, J. , Iborra Pas-

tor, J. L., and Lozano Teruel, J . A. (1982)

J .

Biol. Chem. 257,

8738-8744

24. Rodriguez-Lopez, J. N., Tudela, J., Varon, R., and Garcia-Cano-

vas, F. (1991) Biochim. Biophys. Acta 1 0 7 6 , 3 79 -3 86

25. Wick, M. M. (1979) Cancer Treat.

Rep.

6 3 , 9 9 1 - 9 9 7

26. Jimenez, M., Garcia-Canovas, F., G arcia-Carm ona, F., Tudela,

J., and Iborra , J . L . (1986) Int . J . Biochem. 1 8 , 39-47

27. Serna,

P.,

Rodriguez-Lopez, J. N., Tudela, J.,Varon, R., and

Garcia-Canovas, F.

(1990)

Biochem. J .

2 7 2 , 4 5 9- 46 3

28. Jimenez, M., Garcia-Carm ona, F., Garcia-Canovas, F., Iborra,

J.

L., Lozano, J. A., and Mart inez-Ort iz ,F. (1984)Arch. Biochem.

Biophys. 2 3 5 , 4 3 8- 44 8

29. Garcia-Moreno, M., Rodriguez-Lbpez, J. N., Ma rtinez- Ortiz , F.,

Tudela,

J.,

Varbn, R., and Garcia-Canovas, F. (1991) Arch.

Biochem. Biophys. 2 8 8 , 4 2 7 - 4 3 4

(Lontie,

R.,

ed) , Vol.

11

pp. 207-240, CRC Press , Boca Raton

Biophys.

Res.

C ommun. 4 6 , 8 7 8 - 8 8 4

Acud. Sci.

U .

S.

A.

7 0 , 9 9 3 -9 9 6

30.

Ochiai,

E.

(1973)

Znorg. Nucl. Chem . Lett.

9 , 9 87 -9 91

31. Vanneste, W. H ., and Zuberbuhler, A. (1974) n Molecular Mech-

anisms of Oxygen Activation (Nayaishi, O., ed) pp. 371-404,

Academic Press, New York

32. Ochiai, E. (1985) in Quimica Bioinorgunica,pp. 245-247, Revertb,

Barcelona

33. Duckworth, H. W., and Coleman,

J.

E. (1970) J . Biol. Chem.

34. Gutteridge,

S.,

and Robb,

D.

A. (1975) Eur.

J.

Biochem. 5 4 , 1 0 7 -

116

35. Galindo, J., Pedreiio, E., Garcia-Carmona, F., Garcia-Canovas,

F., Solano, F., and Lozano, J. A. (1983) Znt.

J.

Biochem. 1 5 ,

1455-1461

36. Peiiafiel, R., Galindo, J.

D.,

Solano, F., Pedreiio, E., Iborra, J.

L.,

and Lozano, J . A. (1984)Biochim. Biophys. Acta7 8 8 , 3 2 7 - 3 3 2

37. Cabanes, J., Ga rcia-C arm ona, F., G arcia-Canovas, F., Iborra, J.

L., and Lozano, J. A. (1984) Biochim. Biophys. Acta 7 9 0 , 1 0 1-

107

2 4 5 , 1 61 3 -1 6 25



3810

Kineticechanism

of

Tyrosinase

38. Cabanes, J. , Garcia-Cinovas,

F.,

Tudela, J. , Lozano,

J.

A., and

Garcia-Carmona, F. (1987) Phytochemistry

26,

917-919

39. Tudela, J. , Garcia-Canovas,

F.,

Varbn, R., Garcia-Carmona, F.,

Galvez, J., and Lozano, J . A. (1987) Biochim. Biophys. Acta

9 1 2 ,4 0 8 -4 1 6

40. Tudela, J., Garcia-Canovas, F., Varon, R., Jimenez, M., Garcia-

Carmona, F., and Lozano, J. A. (1987) J . Enz. Inhibit. 2, 47-

56

41. Tudela, J. , Garcia-Canovas, F., Varon, R., Jimenez, M., Garcia-

Carmona,

F.,

and Lozano, J.

A .

(1988)

Biophys.

Chem.

30,

303-310

42. Garcia-Canovas,

F.,

Tudela, J., Martinez-Madrid, C., Varon,

R.,

Garcia-Carmona,

F.,

and Lozano, J. A. (1987) Biochim. Biophys.

Acta 9 1 2 ,4 1 7 -4 2 3

43. Escribano, J., Tudela, J., Garcia-Carmona, F., and Carcia-Cano-

vas, F. (1989) Biochem. J.

262,

597-603

44. Devi, C. C., Tripathi, R.

E.,

andRam aiah, A. (1987) Eur. J.

Biochem. 166,

705-711

45. Tripathi, R. E., Devi, C. C., and Ram aiah , A. (1988) Biochem. J.

252,481-487

46. Devi, C. C., Tripathi, R. E., and Ram aiah , A. (1989) Pigment.

Cell Res.

2,

1-6

47. Bright, H. J., Wood, B. J. B., and Ingraham , L. L. (1963) Ann.

48. Osaki, S 1963) Arch. Biochem. Biophys. 100, 378-384

49. Vaughan, P. F. T., and But t , V. S. (1972) Biochem. J . 1 2 7 , 6 4 1 -

50. Pomerantz,

S.

H., and Murphy ,V. (1974)Arch. Biochem. Biophys.

51. H earing, V. J., and Ekel, J. M. (1976) Biochem.

J.

157, 549-557

52. Cabanes, J., Garcia-Canovas,

F.,

Lozano, J. A,, andGarcia-

Carmona,

F.

(1987)

Biochim. Biophys. Acta 923,

187-195

53. Hartree, E. (1972) Anal. Biochem. 48, 422-427

54. Gerald, C. F. (1978) Applied Numerical Analysis Addisson-Wes-

ley, Reading, MA

55. Thompson, A., Land, E. J. , Chedekel,

M.

R., Subbarao, K . V.,

an d T ru sco t t , T .G. (1985) Biochim. Biophys. Acta 8 4 3 ,4 9 -5 7

56. Marqua rdt, D. W. (1963) J . Soc. Ind. Appl. Math. 11, 431-441

57. Jand el Scientific (1989) SigmaPlot 4 .0 , Jandel Scientific, Corte

58. Cornish-Bowden, A. (1979) in Fundamentals of Enzyme Kinetics,

N .

Y.

Acad. Sci. 180 ,96 5-966

647

160,

73-82

Madera, CA

pp. 34-37, Buttenvorth & Co., Ltd., London

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