Kinetic study on the effect of pH on the melanin biosynthesis pathway

1076(1991)379-386 o 1991FdsevierSciencePublishersB.V.0167-4838/91/$03.50 ADONIS 016748389100109K

379

Biochimica et Biophysica Acta,

BBAPRO33~8

Kinetic study on the effect of pH on the melanin

biosynthesispathway Jos~ N e p t u n o R o d d g u e z - L b p e z t, Jos6 T u d e l a 2, R a m 6 n V a r 6 n t and Francisco Garcia-Cfinovas 2 I C,~tedeade Qulmica Itwhcctrial, E.U. Polit~cnica, Universidad de Castilla.La Mancha, AIbacete and z Departamento de Bioqulmica y Biologia Molecular. Facultad de Biologla, Universidad de Marcia, Espinardo, Murcia (Spain)

(Received11 June 1990) Key words: Melanosenesis;Melaninbiosynthesis;Tyrosinase;L-Dopa;Enzymekinetics This paper deals with the quantitative description of the regulatory effect of pH on the oxidatien pathway of L-4IOIMItO yield mdanin~. Tyroslnase catalyzes the oxidation by molecular oxygen of L-dopa go o-dopalplincMl~ which ~olv4~ mm-enzynmtically through a branched pathway with eydizafion or hydroxylation reaetlons. The woduction of several qulnones and semiq~nenes in the pathway has also been reported. The intermediates of the hydxoxylation branch have been identified and the conesponding rate constants have been determined. These compmmds, such as have been detected in melanosolnes and in tmnoral cells, have great cytotoxic power and could have physiological signWtcanee in acidic media. Introduction Tyrosinase (EC 1.14.18.1, monophenol, r-dopa: oxygen oxidoreductase) is a copper protein widely distributed in the phylogenetic scale [1-2]. It has two different catalytic functions: (a) monopbenulase, producing the hydroxylation of monophenols (tyrosine) to o-diphenols (dopa) and (b) diphenolase, oxidiTJng o-diphenols (dopa) to o-quinones (o-dopaquinone). The products generated by this enzyme, the o-quinones, are very reactive and tend to stabilize through intramolecular [3] or intermolecular reactions [4]. They give, depending on the substrate (o-diphenuls) and on the medium conditions, different reaction products [5]. Dopa has been shown to be selectively toxic to human pigmented melanoma cells 'in vivo" [6]. The cytotoxicity of this compound has been attributed to its selective uptake by melanocytic cells [6] and to the Abbreviations:dopa. L-3,4-dihydroxyphenylal~nlne;topa, L-2,4,5-trihydroxyphenylalanine; o-dopaquinone, 4-(2-carboxy-2-aminoethyi)1,2*benzoquinone; o-topaquinone.5-(2-carboxy-2-aminoethyl)-4-hydroxy-l,2-~uinone; p-topaqainone,5-(2-carbory-2-aminoethyl)2-hydroxy-l.4-benzoquinone:dopachrome, 2-enrboxy-2,3-dihydrolndole-5,6-quinone; leukodopachrome, 2,3-dihydro-5,6-dihydroxyindole-2-catboxylate, Correspondence:F. Garc|a-CAnovas,Departamentode Bioquimieay Biologia Molecular, Facullad de Biologia, Univetsidadde Murcia, E-30100Espinardo,Murcia,Spain.

formation of reactive quinones and semiquinones formed in situ during metabolic activation of this compound by the enzyme tyrosinase [6-8]. The triphenolic amino acid topa also shows a selective toxicity to melanoma cells [9] which is much higher than that of the natural melanin precursor dopa [6], although the general toxicity of topa has precluded its use in melanome therapy. The cytotoxic properties of topa seem to be due to the susceptibility of this substance to oxidation [10]. It is possible that the cytotoxicity is mediated by oxygen radical or H202 formed at oxygenation of topa [11]. Another explanation of the cytotoxicity of topa could be the reaction of the quinone formed by oxidation of topa with nucleophilic groups of cellular macromolecules [7,9,12,13]. Tyrosinase catalyzes the oxidation of dopa [14,15] by molecular oxygen to yield o-dopaqulnone, which evolves non-enzymatically through a branched pathway consisting of cyclization or hydroxylation reactions (Fig. 1). Semiquinone production may be significant in the presence of spin-stabilizlng metal ions [8]. This pathway has been partially characterized at pH values where only the intermediates of the cyclization branch have been identiffed, and the corresponding rate constants determined. The aim of this paper is to obtain the overall characterization of the oxidation pathway of dopa catalyzed by tyrosinase. The experimental study attempts to identify the intermediates of the hydroxylation branch and to determine the corresponding rate constants. This

380 SlOW MELANINS"

.o~coo" HO"A''~

slow ii1~.-. DC 9

E ~

/

t.-I

o%.,~.,-yCOO" --7

°~r~.r~coo"

~a i,. 0 . ~

Nla~

k,.." oyy.ccoo"~

k.,

O'~'~s

O

:p " 0 " ~

k, -.

"Ov~,,"yCO0-

,,. 2o.1~._7j_~

I~'12

HO'~"~"-'~I"

0

L

"

H2

"tlO°

;' Fig. 1. Pathwayproposedfor the oxidationof dopa catalyzed by I,/rosinaze,takinginto considera!ic,n the effect of pH. E = tyros.nase;D = dopa; QH = o-dopaquinone-H+; Q = o = dopaquinone; OSQ = o-dopasemiquinone; L = leukodopachrome; T = tnpa, FQ = p-topacluinone; PSQ= p:.)pasenfiquinone;and DC = dopachrome.

allows the quantitative description of the regula,*ory effect of the pH on this pathway. The intermediates ~f the hydroxylation branch, such as those detected in melanosomes [16-18] and in tumoral cells [19,20], have great cytotoxic power [21], and coul~ have physiological significance in acidic media. Materials and Methods

Materials Mushroom tyrosinase (3300 units/rag) and dopa were purchased from Sigma (U.S..~.). All other chen'tical3 were of analytical grade, suppfi~:l by Merck (F.1LG.).

mediL:m was ~ mM acetate buffer with 100 mM NO3K in order to obtain a constant ionic strength. Saturation conditions for dopa at pH 6.0 were obteined at 2.0 ~ M [23]. Other reagents and conditions are detailed ia ~he iegeads of ~he figares. Oxygen determination. Oxygen consumption was fob lowed by a Hansatech DW oxymeter, based on the Clark electrode Temperature was con trolled at 17 o C using a H etofrig circulating bath with a heater/cooler and chect'~d using a Cole-Panther digital ~hermometer with an SR +_0.1°C. Protvin concentration was determm, ed by a modified L~wry method [24].

Methods Rapid-scan assays. Spectrophotometric measurements

Results gral Discussioe~

were carried out with a Perkin-Elmer Lambda-2 spectrophotometer interfaced on line with an AMSTRAD PC2086 computer. The scan speed was 20 n m , s -], and the first recording was started at 20 s from the beg/nning of the reaction. The assay medium contained 25 mM acetate buffer (pH 4.3) and other conditions are detailed in the legends of the figures. Matrix analysis of the iterative spectra was carried out by application of the test for two or three absorbing species in solution, with stoichiometric restrictions [22]. Kinetic assays. Product accumulation was spectrophotometrically followed at 475 nm using the same instrument as for rapid-scan assays. The reaction

Rapid-scan assays In an attempt to study the role of pH on the melanin hiosy~hesis pathway, dopa was oxidized in the presence of tyrosinase and NaIO4.

Oxidation cat¢d~,zedby tyrosinase The iterative spectrum for oxidation of dopa at acid pH (Fig. 2A) showed the initial formation of an absorbaace maximum at 390 ran, and the further appearance of another maximum at 480 nm. The high enzyme concentration usec~ caused rapid oxygen exhaustion, characterized by crossing between tracings, but with no clearly defined isosbestic point. The graphi-

381

A

0.3 A

";:::: . . . . . . ::-:::-;:". • 7-.~~" ~. . . . .

O.1 ~

~.."--~;:,':Tt

'

'"'~" ""::~

I

350

/-,50 1l-

-

-_.:

Mnm

B

..

.

550

Under acid pH conditions (Fig. 2A and B) the intermediates of both the cyclization and the hydroxylation branches of Fig. 1 are present. Thus, the three absorbing species detected by the matrix analysis could be o-dopaquL=one (Zmax = 390 rim), dopachrome (Zm~ = 475 nm) and p-topaquinone (~m~ = 485 rim [25]). The non-detection of the regenerated dopa, leukodopachrome, as well as o- and p-semiquinones is due to the same reasons as those described for the cyclization branch. The hydroxylation branch, however, involves the acct:mulation of another intermediate, as can be derived flora the non-appearance of an isosbestic point (Fig. 2A). This coml:ound might be topa, which is not an at~sorbing species in the visible range.

Oxidation by periodate It is well known that sodium periodate oxid~¢s a-diphenols to their corresFonding o-quinones [25]. When [dopa] was greater than [NalO4] at acid pH (Fig. 3A) tl~¢ iterative spectrum was similar to that carried out with high tyrosinase concentrations (Fig. 2A). "l'he pres-

----

< I

<

0

A

A

-0.4

-0.2 Amj- A~j./Ani'A.

i•

Fig. 2. Rapid-sennassays for the oxida0oil of dopa catalyzed by iyrosinase. Reagents: 1 mM dop~ 0.13 mM 02 and 0.20 mg/ml iyroshiase. Other conditions as detailed in Materials and Methods. (A) Iterative spectrum. (B) Matrix analysis of the spectrum. A,~absorbance value 'it wavelength i and tracing j. m = 389 rim; n = 455 rim; and i (rim)= 364 (o), 376 (r3). 429 (~). 475 t l ) , 508 (t); j" = third recording.

0.1 ."- .-:~s.-~,, . . . . .

,

l~J"--:7;. ;.'

"

350 ca] analysis of these recordings by the rank matrix method [22] fulfills the test for a minimum of three absorbing species in solution with stoichiometric restrictions (Fig. 2B). At greater pH values [15], absorption maxima at 390 and 475 n m were obtained, one isosbestic point was defined at 416 nm, and at least two absorbing species were detected by the matrix analysis. These data, in accordance with other experimental results [15], enabled the two absorbing species (Fig. 1) to be identified as o-dopaquinone (;%ma~= 390 nm) and dopachrome (;~m.~ = 475 rim). Leukodopachrome was not accumulated in die assay medium, as waz revealed by the appearance of one isosbestic point. The non-accumulation of o-semlquinones is due to the absence of spin-stabilizing metal ions [8]. In addition, the formation of dopachrome was simultaneous with the regeneration of dopa (Fig. 1), for wliich reason the o-diphenol was not con;idered as one linearly independent substance by the matrix analysis [22].

",.." ~\~

--. " - ~

450

k (rim)

B

1 c ~1( ~.~

~-

550

--

--=

-

-

•

.:.

<-0 -0. 35

Amj<. A m j , / A n ~ . A n j .

- 0.25

Fi 8. 3. Rapid-scan assays for the oxidation of dopa by a deficit of N~IO4. Reagents: ! .0 m M dopa and 0.1 m M NalO=. Other conditions as detailed in Materials and Methods. (A) lterativ¢ Sl~Ctruin. (B) Matr~x analysis of the spectrum, n= = 3.03 , .n, n = 456 nm; i ( n m ) = 361 (El), 371 (o), 424 (~.), 47"1 C~i), 531 (O); j ' = first recording.

382

A

0.8 A .. ~ ; . ~ : - ~

---

- - - - - _

-

. - ~ ....

0.4

I

350

450

550

~(nm)

0.6

-,-,

03

o

0

Ai.j ° Ai,j.

0.5

Fig. 4. Rapid-scan assays for the oxidation of dopa by a excess of NalO4. Reagents: 0.4 mM dopa and 4.0 mM NalO4. Other conditions as detailed in Materials and Methods. (A) Iterative spectrum. (B) Matrix analysis of the spectrum, i ' = 450 nm; i (nm) = 370 (o), 377 (O). 430 (&), 470 (A), 510 (n), 530 (o); j ' = first recording.

ence of at least three absorbing species in solution was detected by matrix analysis (Fig. 3B). At higher pH values [15], a great similarity with the respective enzymatic assays (described in the above section) was also obtained. When [NalO4] was greater than [dopa] at acid pH, a set of recordings was obtained which showed the presence of two absorbance maxima at 390 and 465 nm, as well as an isosestic point at 391 nm (Fig. 4A). The existence of a minimum of two absorbing species in solutions was detected by matrix analysis [22], since a set of straight lines intersecting on the origin of the coordinates was obtained (Fig. 4B). Similar results were reported at greater pH values [15], but the isosbestic point appeared at 398 nm. The periodate anion might act as oxidant in the following reactions of the pathway depicted in Fig. 1: dopa --, o-dopaqulnone, leukodopachrome --, dopachrome and topa--* o-topaquinone. The non-accumulation of o-semiquinones in these experiments (Figs. 3A and 4A) is also due to the absence of spin-stabilizing metal ions [8]. When [NalO4] is lower than [dopa] the

periodate anion is exhausted in the stoichiometric formation of o-dopaquinone (Fig. 3A), in a parallel way to the oxygen depletion catalyzed by tyrosinase (Fig. 2A). The similarity between the tyrosinase and periodate assays supports the non-enzymatic breakdown of odopaquinone (Fig. 1), at acid pH (Figs. 2 and 3) and greater pH values [15]. When [NaIO4] is higher than [dopa], a stoichiometric quantity of periodate is consumed in the formation of o-dopaquinone (Fig. 1), which suffers non-enzymatic cyclization or hydroxylation to yield leukodopachrome or topa, res~aectively. These intermediates are not accumulated m solution, since they are also oxidized by the excess of periodate to provide the corresponding dopachrome or o-topaquinone ()Lm~ =465 run [25]). The non-accumulation of the intermediates enables the appearance of an isosbestic point, whose hipsochromic displacement at acid pH (Fig. 4A) is due to the operation of both branches of the pathway (Fig. 1). Under these conditions, the matrix analysis detects at least two absorbing species, o-dopaquinone and dopachrome/otopaquinone. The products are generated in a constant ratio during the entire assay time, determined by the cyclization and the hydroxylation steps, respectively. Thus, one of the two compounds is considered as linearly dependent on the other by the matrix analysis [22]. Thus, the above results with tyrosinase and periodate, as well as data obtained with cyclic voltammetry assays [15] support the reliability of the oxidation pathway of dopa proposed here (Fig. 1). Kinetic

assays

The reaction progress of dopa oxidation catalyzed by tyrosinase was followed by measuring the appearance of

0

150

t(s)

300

Fig. 5, Kinetic assays of product accumulation for the oxidation of dopa catalyzed by tyrosinase. Reagents: 2 mM dopa and 0.26 mM O 2 at pH 4.3. Other conditions as detailed in Materials and Methods. (A) Spectrophotometric recording. Tyrosinase (~g/ml): 6.6, 13.3, 20.0, 26.6, 33.3. (B, O) Corresponding values of the lag period (~-) vs. enzyme concentration (E0). (n, A) Plot of products accumulation rate in steady-state conditions against Eo.

383 products (A475) during the entire assay time. The experimental recording (Fig. 5A) presents a marked lag period (~). At acid pH the lag period decreased when the enzyme concentration rose (Fig. 5B), whereas no dependenee between them was obtained at greater pH values [14]. This kinetic behaviour has not yet been analyzed and could be useful for the quantitative characterization of the pathway. The absolute values of the steady-state rate of the enzymatic step have been determined by measuring oxygen consumption (Fig. 6). This is due to the presence of two absorbing products at 475 nm, whose relative proportions are not known. These rate values are one-half of that of o-dopaquinone-H ÷ formation (Fig. 1), since one molecule of oxygen generates two molecules of quinone in each turnover [26].

I

A

r;o

K,

~

Melan!ns (sl°w) J 'D~"-~ ~/~i

A

301Eo(Pg/ml } 60 t(s}

0

120

Fig. 6. (A) Kinetic assays of oxygen consumption for the oxidation of dopa catalyzed by tyrosinase. Reagents: 2.0 mM dopa, 0.26 mM O2 and tyrosinase (pg/ml): 6.6, 13.3, 20.0, 26.6, 33.3, 40.0 at pH 4.3. (B)

Correspondingvaluesof Vovs. E0.

Kinetic analysis The oxidation pathway of dopa catalyzed by tyrosinase at acid pH (Fig. I) can be depicted as follows: 2 PSQ

,/I "<

~=_o.1

steady-state of the pathway, however, it is possible to derive explicit equation~ relating the product concentration o, with the assay time. Kinetic analysis of this pathway requires the consumption of dopa as well as the breakdown of the products to be negligible during the reaction time. Both conditions have been verified in the experimental assays. The mass balance between the reagents (Scheme I) states that the consumed o-dipbenol (V0t) yields intermediates (Z[I]), products ([DC] and [PQ]) and regenerated o-diphenol, through the cyclization and the hydroxylation branches of the pathway: Yot + ks[D][QH]t + k_3[D][DC]t + k_,[DI[PQ]t - k_s[OSQ]2t -

k4[T][QH]t

-

k3[QH][Llt ~[tl+ [VCl+ [~1 =

~[I] ffi[QH| + [QI+ [L] + iOSQl+ [PSQI+ [TI

(1) (2)

The generation and breakdown steps of semiquinones are in rapid equifibrium [8]: Scheme I

This scheme involves the steady-state rate of the enzymatic step (V0), first- (k I and kc) and second( k _ l , k2, k3, k_3, k 4, k_4, ks, k_5, k 6 and k_e) order rate constants. The experimental results at acid pH (Figs. 2A and 3A) show the accumulation of topa (Scheme I). This implies a significant contribution of the second-order steps controlled by k 4 and k-4, to the kinetic behaviour of the pathway. Thus, there are no analytical solutions describing the evolution of the products during the tran_~;.~t phase of kinetic assays (Fig. 5A). At the

ks[DI|QH] = k_s{OSQI2, k6[TI[I'QIffik_6[PSQi2

(3)

There are reversible steps displaced towards the regeneration of dopa [14]: ksIQHI(LI ~ k_~IDCI[D], k,IQHll T] > k-4IPQ]ID!

(4)

Furthermore, the contribution of L, OSQ and PSQ to Eqn. 2 is negfigible, since these intermediates are not accumulated during the assays (see Rapid-scan section). Therefore, the net formation of PQ is equivalent to

384 k4[T][QH]t, whereas the net production of DC is determined by kc[Q]t and Eqn. 1 is transformed in: 2([DCI + [PQI) =vot -.~[q

Furthermore, since Vo increases when pH is raised, a negligible contribution of the second quotient as regards the first leads to:

(5)

¢ = (k_ ~[H+ ! + kc)/2k:kc During the steady-state of the pathway, the concentration of the intermediates remains constant: [QI:I] ffiVo + k_ I[H + ][Q] - kl[Qa ] - k~[Ql - k~[QH] -

(12)

an expression not dependent on the enzyme concentration. On the other hand, Eqn. 10 can be rearranged as:

k4[QHI[T! =0 ¢ = 2a + fi(l/Vo)

[Q] = kl[QHI - ( k _ ~[H + ] + kc)[Q] = 0

(13)

(6)

where: ['[1 = k~[QH]- k~IQH][T] =

0

where k~ = k2[H20 ]. From the above system of equations, expressions for the concentration of the intermediates at the steady-state are obtained: [QH].

[Q]~

( k-ilH+ l + kc)Vo 2klk~+2k~(k_l[H+]+k~)

k~Vo 2klkc+2k'~(k-ilH ÷]+kc)

1 + kcKa ~ [ n l+k--~ 2a= [H÷ [ + ~_~2K a

04)

and p = k~/t, 4

(7)

(15)

K a = k l / k _ 1. These expressions are useful to determine the rate constants of the pathway.

0% = k~/k4

The consideration of Eqns. 1, 2 and 7 leads to the expression: Vo[ [ kl+k_l[H+]+k~ k~ 11 [DC]~+[PQ]~ = ,-~--[,-[ 2klk ¢ +2k~(k_l[H+]+k¢) + k--~oo]] (8) Thus, the accumulation of the products of the pathway during the steady-state follows a straight line (Eqn. 8), whose intercept with the time axis defines the lag period

0"):

kl + k_l[H+]+ k¢ k~) ffi 2klk ¢ +2k~(k_l[H + ]+ k©) +

(9)

Since k c :~ k 1 [14], the previous equation becomes: k_llH+l+kc k~ T= 2klkc + 2k ~( k_ :[H+ ]+ kc ) "~ k4Vo

(10)

This equation predicts that • decreases when the enzyme concentration ( E 0, involved in Vo) is raised, according t.o experimental data at acid p H (Fig. 5). At $reater pH values, however, 7 is not dependent on the enzyme concentration [14]. Thus, when [H + ] decreases: 2k~'(k_l[H + ] + kc) < k l k ¢, (k~' << kt) and Eqn. 10 is transformed into: k_dH+]+k~

k~

, = 2---Tk~-7--k + (-~ff~%

(n)

Kinetic data analysis Two series of spectrophotometric (Fig. 5) and electrometrie (Fig. 6) assays were carried out by using the same set of enzyme concentrations. From A475 vs. t recordings, data of the final portion were fitted by linear regression and the corresponding ¢ vs. Eo values were calculated. The initial zone of the [02] vs. t recording were fitted by finear regression, yielding the respective values of Vo/2 vs. E o. The kinetic assays, therefore, provide ~- vs. (1/Vo) data at the same E o values. This procedure was applied to several pH values (Fig. 7A) and the data fitted by linear regression according to Eqn. 13. The great similarry between the slopes of the straight lines (Fig. 7A) confirms the non.dependence of fl on pH (Eqn. 15). Thus, the mean values of the slopes enable k ~ / k 4 to be calculated (Fig. 713). The intercepts on the ordinate axis, however, show a hyperbolic behaviour on [H +] (Fig. 7B). The non-linear regression fitting of 2 a vs. [H +] data to Eqn. (14) leads to the determination of all the rate constants of the pathway, by considering K s = 1.91 nM [27]. Initial estimations of k 1, k~ and k c are obtained, respectively, from the lower limit, higher limit and half-saturation value of the hyperbola (Fig. 7B). The values of the rate constants of the pathway (Fig. 1 and Scheme I) are described in Table I. Effect of the p H on the melanin biosynthesis pathway The spectral and the kinetic assays described above support and characterize the oxidation pathway of dopa proposed iler¢ (Fig. 1). The pH of the medium de-

385 termines the differential contribution of the cyclization and the hydroxylation branches, through the protonation/deprotonation of the key intermediate, o-dopaquinone. Thus, the above experimental data show that both branches are only significant, simultaneously, at

TABLE !

Values of the rate constants for the oxidation pathway ~f dopa catalyzed by tyrosinase (Fig. I), at 17°C Constant (units)

Value

k I (s -1) k2 (M-I's -I) kc (s - l ) k4 ( M - L s - I )

O.OS+ 0.01 (1.01+ 0.03)-10 -4 65.3 +12A (1.62:t: 0.22).103

"C(s) 100

50 ~ ' - " ^ " ~ ' ~ e - °o

0

0 1

"2

o

9 t

18

I/Vo (pM4. s)

150~1 3

00

B

130

acid pH. Indeed, there is evidence concerning the acidic character of melanosomes [16-18] and tumoraI ceils [19,201. The cytotoxicity of dopa and other melanogenic eatecholamines has been attributed to further products of oxidation [6,7]. Compounds similar to the intermediates of the hydroxylation branch also have cytotoxic power [21], with greater selectivity than intermediates of the cyclization branch [9] on melanome cells. The quinonic intermediates show high reactivity with amino and thiol groups of amino acids and proteins, inorganic anions and reductant agents, processes which could be related with their cytotoxic ability [1,5]. The appearance of semiquinones might be increased under physiological conditions, due to the presence of spin-stabilizing ions (Cu 2+, Fe 3+, Zn2+). These ions have been found in a variety of melanised structures [28], including the choroid of the eye [29-30], black hair [31] and isolated melanosomes from Harding Passey, horse and human melanomas [32]. The reaction between semiquinones and snlfydryl amino acids~ such as cysteine, is likely to trigger a cascade of free radical reactions. Thus, increased production of cysteinyldopas has been found during dopa therapy [33]. Therefore, the production of even small amounts of semiquinones in the pathway of oxidation of eatechols and eateeholandnes may have toxicological significance [8]. Therefore, reagents causing changes in the pH of the physiological microenvironment, could determine the actual cytotoxicity of dopa and other eateeholamines. Acknowledgements

[H'] (yM)

Fig. 7. Kinetic m~saysof the oxidation of dopa catalyzed by tyrosinase, at different pH values for several enzyme concentrations. Values of pH: 3.9, 4.0, 4.2, 4.3, 4.5, 4.6, 4.8, 4.9, 5.2, 5.4, 5.5, 5.9; tyrosinase Otg/ml) for each pH: 6.6. tO.O, 13.3. 20.0, 26.6, 33.3; other assay conditions as detailed in Materials and Methods. (A) Dependence of the lag period (v) on l/V0, kinetic parameters obtained from spectrophotometric and oxymetric recording, respectively. Experimental data and linear regression fittings are shown. (B) Corr~ponding values of

This paper has been partially supported by a grant from the Comisi6n lnterministerial de Ciencia y Tecnologla (CICYT), :~'ojeet number AL189-674. J.N. Rodrlguez-Lbpez has received a fellowship from the Comunidad Autbnoma de Castilla-La Mancha, Spain.

References

2a and of 0 vs. [H÷ ]. (O, A) experimentaldata of 2a and of 0, respectively. (O-----4D) data calculated by using Eqn, 14, with the initial estimations of their kinetic constants. (Q 0) data calculated by using Eqn. 14, with the final estimations of their kinetic constants, obtained from non-linear regression fitting. (& A) mean value of ~ vs. [H + l, according to Eqn. 15.

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