The catalytic effect of tyrosinase upon oxidation of 2-hydroxyestradiol in presence of catechol

ARCHIVESOFBIOCHEMISTRYAND BIOPHYSICS Vol. 232, No. 1, July, pp. 189-196, 1984

The Catalytic Effect of Tyrosinase upon Oxidation of 2-Hydroxyestradiol in Presence of Catechol GERT M. JACOBSOHN’

AND MYRA

K. JACOBSOHN*

Department of Biological Chemistry, Hahnemann University, Phikddphia, Pennsylvania lglE2 and *Department of Biology, Beaver Cdlege, Glenside, Pennsylvania 19058 Received October 17, 1983, and in revised form March 9, 1984

The hydroxylating activity of mushroom tyrosinase has been utilized for over a decade in the preparation of 2-hydroxyestradiol from estradiol, yet this same enzyme is known to function as an oxidant of o-dihydric compounds to the corresponding oquinones. It was questioned why catechol estrogens do not react further, particularly since the tyrosinase activity (hydroxylating) is exceeded many fold by the diphenol oxidase activity of the enzyme. This report describes that the catechol estrogen will react in presence of enzyme but only if catechol is also present. Diphenol oxidase activity was measured either by the polarographic oxygen-utilization technique or by changes in the absorption spectrum at 206 and 256 nm. The enzyme activity was standardized with catechol (Km = 5.2 X lop4 M). The steroid did not react with the enzyme if catechol was absent. With catechol, the steroid reacted rapidly and completely (Km = 4.2 X 10e4 M). The consumption of oxygen with catechol and 2-hydroxyestradiol was additive and stoichiometric, 1 g-atom oxygen/m01 of either substrate. Kinetic analysis shows that catechol functions as an activator of the tyrosinase.

Mushroom tyrosinase, EC 1.14.18.1, has been used for some time in the preparation of 2-OH Eg (Fig. 1) from estradiol (1) by the procedure of Jellinck and Brown (2). The oxidation, which is dependent upon molecular oxygen, is performed with an excess of NADH as hydrogen donor and stops with the introduction of the second hydroxyl group into the aromatic ring of the steroid. Yet the enzyme is known to perform two roles: (i) the hydroxylation of monophenols to their dihydroxy derivatives, and (ii) the oxidation of the latter to the corresponding o-quinones. These effects have been referred to as the cresolase and catecholase (diphenol oxidase) activities, respectively, indicating the preferred 1To whom correspondence should be addressed. 2Abbreviations used: 2-OH El, 1,3,5(10)-Estratriene-2,3,17j3-triol; Bicine, N,N-bis(2-hydroxyethyl)glycine.

substrates for standardizing the enzyme. We attempted to measure the latter activity of the enzyme on 2-OH EZ and found that it was nil, unless catechol was added to the incubation medium. With catechol present, the steroid reacted rapidly and completely. The consumption of oxygen with catechol and 2-OH Ez was additive and stoichiometric, 1 g-atom oxygen/mol of substrate. In mammalian systems, tyrosinase is responsible for the production of melanin, and is the only enzyme involved in its formation (3). The enzyme accumulates in melanosomes, a specialized cytoplasmic particle of melanocytes, and is transferred to these from smaller particles isolated as microsomes (4). The reaction proceeds by hydroxylation of tyrosine to DOPA, oxidation to DOPA quinone, and nonenzymatic polymerization to melanin. Recently, it was reported that 5,6-dihydroxyindole 189

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190

JACOBSOHN

AND

JACOBSOHN

OH

FIG. 1. BHydroxyestradiol.

RESULTS

By polarographic means it was found that 2-hydroxyestradiol was unreactive to tyrosinase but, if catechol was also present, the steroid reacted rapidly and completely (Fig. 2). If catechol was added first, the steroid reacted and caused the consumption of oxygen which, on a molar basis of steroid, was equal to the oxygen loss caused

can be converted to melanochrome by a preparation from murine melanomas and by the skins of brown mice, using DOPA as cofactor (5). A parallel reaction appears to occur with 2-OH Es but the mechanism of control through an activator is different. MATERIALS

AND

2.HYDROXYESTRADIOL

METHODS

Mushroom tyrosinase (Sigma, 2236 U/mg of activity with L-tyrosine as substrate) was used at a concentration of 14 mg/liter of incubation medium. 2-Hydroxyestradiol was supplied by Steraloids (Wilton, N. H.) and gave a single spot on chromatograms in the system described below. It was dissolved in methanol, and aliquots not exceeding 15 pl were used in each incubate. The buffer consisted of 0.05 M KHzPO,NaOH at pH 7.0. Catechol was of the highest available reagent grade. The assay consisted of either measuring the oxygen consumed polarimetrically with a Clark electrode in the Gilson K-IC Oxygraph or measuring changes in the absorption spectrum of the incubation mixture at 206 and 256 nm in an Aminco DW-2 Spectrophotometer. The buffers, 1.8 ml for polarimetric and 2.7 ml for spectrophotometric assays, were placed in cells with 25 or 37.5 pl of a solution of the enzyme in the same buffer. For polarimetric measurements, the appropriate amount of catechol dissolved in buffer was added first, then the steroid, for spectrophotometric measurements, the order of addition of the two substrates was reversed. All incubations were performed at 23°C. The spent incubation media were adjusted to pH 3.5 and extracted once with an equal volume and once with one-half their volume of either a mixture of methylene chloride-ethyl acetate, 21 (v/v) or with benzene. The pooled extracts were dried over Na.#O, and the solvents were removed with nitrogen jets. The residues were dissolved in methanol, applied to thin-layer chromatography plates (Silica Gel H, 250 pm thick, Applied Science) and run in the system cyclohexane-ethyl acetate-ethanol, 1091 (v/v) (6). Steroids were made visible by exposing the plates to an atmosphere saturated with iodine vapors or by spraying with 50% aqueous sulfuric acid and heating to 106°C for 5 min.

TIME

(secl

TIME

bed

B

I

FIG. 2. Polarographic curves of oxygen consumption produced by (A) adding 2-hydroxyestradiol first, then catechol, and (B) adding eatechol first, then 2-hydroxyestradiol. Total volume, 1.8 ml. Final concentrations: catechol, 2 X lo-’ M; 2-hydroxyestradiol, 2 X 10 -’ M; tyrosinase, 14 mg/liter; KHaPO,-NaOH buffer at pH ‘7.0.0.05 M; Temperature, 23°C.

TYROSINASE

EFFECT

UPON

by an equivalent amount of catechol. If the steroid was added first, oxygen consumption could not be detected. Once catechol was added to the steroid-containing mixture, a decrease occurred in the concentration of oxygen which was equal to the sum of its consumption with catechol added first and its consumption with the steroid added second. Based upon values of oxygen content of air-saturated water calculated by Estabrook (7), the total consumption of oxygen in nanoatoms equalled the amount of catechol in nanomoles incubated, with an error less than 10% of the calculated value. Catechol was subsequently used as an internal standard to relate polarographic curves to oxygen consumption with catechol estrogen as substrate. A doublereciprocal plot for rates of oxygen consumption with catechol as substrate gave a K, of 5.2 X 10h4M with a V,, of 1.9 X 10e5 M SE!C-l.

The initial rates of oxygen consumption at fixed concentrations of steroid but variable concentrations of catechol are shown in Fig. 3. Oxygen consumption rates increased with higher levels of catechol up to about 1 X 10e4 M concentration of the latter, at which point the curve started to level off. Lineweaver-Burk plots for 2-OH Ez showed no important differences when calculated from data obtained with variable catechol concentrations from 1.2 X 10d4to 2.0 X 10e4M, and these data were averaged. The Km for 2-OH Ez, in the presence of catechol, was 4.2 X 10e4M with a

191

2-HYDROXYESTRADIOL

4.0

3.0

2.0

0 i

00

4.0

6.0

12

16

20

0.6

1.2

1.6

2.0

B m 3p x 1.6 -

A.

'p D

l.O-

Ji

0 0

0.4

105 X [Catecholl

Wf)

FIG. 3. Initial oxygen-consumption rates at fixed concentrations of 2-hydroxyestradiol(2 X lo-” M) and increasing amounts of catechol. (A) Graphic expansion of (B). Other conditions as in Fig. 2.

dition of the last reagent and, 2 min after start of the reaction, only a shoulder remained. The final reaction product yielded no distinct spectrum in the near-ultraviolet region, but showed three shoulders, at 265,285, and 322 nm (not shown in Fig. 4). A broad band around 225 nm was also v,,, Of 1.2 X 1oe5 M SeC-‘. present. The trough noted at 256 nm rose rapidly Absorption spectra between 200 and 350 nm were measured for substrates and during enzymatic reaction; the absorbance products of the reaction (Fig. 4). The vol- at 206 nm remained relatively constant. ume of incubation medium was 2.7 ml These two wavelengths could be used for rather than 1.8 ml as in the oxygen-utithe determination of the rate of enzyme lization measurements, but the concentra- activity by the dual-wavelength method or tions of enzyme and reagents were kept the difference in light transmission beconstant for the two types of assays. Peaks tween these two points (Fig. 5). All specfor catechol at 2’77 nm (E = 2030) and for trophotometric assays were performed in 2-OH Ez at 288 nm (E = 3730) were noted. this manner. The rate of reaction with time Addition of catechol to a steroid-containing is nonlinear, whether measured by differenzyme mixture caused an initial hypso- ences in transmittance or absorbance. Even chromic shift of the 288-nm band with an after 25 s there is a slow increase in abincrease in absorbance, no doubt due to the sorbance, perhaps indicative of the colored additive effect of both bands. This peak pigment formation noted below. By specbegan to decrease immediately upon ad- trophotometry it was found that the initial

192

JACOBSOHN AND JACOBSOHN STEROID PLUS ENZYME CATECHOL, ZYWLATER

PLUS

STEROID PLUS ENZYME PL “S CA TECHOL

200

221

250

277 WAVELENGTH

288

300

350

fnm)

FIG. 4. Spectral changes of E-hydroxyestradiol between 206 and 350 nm induced by tyrosinase action with and without catechol. Total volume, 2.7 ml. Other conditions as in Fig. 2. Scanning speed, 5 rim/s; Absorbance, 1.0; Slit opening, 3 nm. Discontinuous lines below 240 nm indicate increased attenuation to absorbance 2.0.

velocities of the reaction were linear with increases in catechol concentration (Fig. 6). Concentrations of catechol between 0.5 X 10e4and 2.0 X 10e4M were directly related to the rate. This is in contrast to the oxygen consumption trials, Fig. 3, where concentrations of catechol above 1.0 X 10e4M showed only minor increases in speed.

15SECI /./

In the absence of catechol and after 25 min of incubation time with tyrosinase, the peak of 2-OH E2 at 288 nm shifted to 285 nm and decreased in intensity by 4%. The trough at 256 nm also increased (Fig. 5). The shifts, along with an increase in the baseline at 320 nm, are in the same direction but much weaker than the shifts ob-

CONDITIONS: 2.7 mL KH,PO,-NaOH buffer, 0.05 M, pH 7.0 2 X 1Om4M P-hydroxyestradiol (1) Add 37.5 ~9 tyrosinase (14 mg/L) (2) Add catechol to 1 X 10-4M Temperature, 23O C Slit, 5.0 nm; recorder speed, 1 in/5 sec.

TIME

(set)

FIG. 5. Dual-wavelength spectrum at 266 and 256 nm of 2-hydroxyestradiol reacting in presence of catechol and of tyrosinase. Conditions as in Fig. 4. Upper lines are continuation of lower lines.

TYROSINASE

EFFECT

UPON

193

L-HYDROXYESTRADIOL TABLE

I

CHANGESINLIGHTABSORFTIONOF %HYDROXYESTRADIOLINTHE PRESENCEOFTYROSINASE %ax bm)

c

%in bm)

Initial

288

3730

256

810

Final”

232 282 324

11340 5760 4500

225 264 307

11160 5310 4230

’ After

c

108 min.

2.0

On

”

0.5

1.0 lo4

1.5 X [Catecholl

2.0

2.5

(Ml

FIG. 6. Initial rates of reaction of 2-hydroxyestadiol in the presence of increasing concentrations of catecho1 measured by dual-wavelenth spectra at 206 and 256 nm. Conditions as in Fig. 4.

served with added catechol. This small rate of reaction is ascribed to residual catechol or catechol-like substances present in the enzyme preparation or activity of the enzyme without the benefit of catechol. The low activity of enzyme on 2-OH Ez determined spectrophotometrically could not be detected in the polarimeter due to insufficient sensitivity of the latter method. To ascertain the effects of tyrosinase upon 2-OH Ez in the absence of catechol, the temperature of reaction was raised to 37°C and the time was extended to 108 min. Spectral changes were insignificant beyond that time. Table I indicates changes in the spectrum. It is noteworthy that the initial change in peak location from 288 to 285 nm, accompanied by a small decrease in intensity, was superseded by a further hypsochromic shift and an increase in absorbance. However, the trough noted at 256 nm broadened and increased in intensity to a larger extent than the peak. The net peak height, measured from the trough, was less after reaction than before. The data show the possible presence of a variety of products. By dual-wavelength spectrophotometry it was found that a catechol

concentration as low as 2 X 10e5M caused a 20-fold increase in the rate of reaction of 2-OH Ez compared to the rate in the absence of catechol (Table II). Visual inspection of reaction products showed formation of a reddish-tan-colored pigment which became deeper in color for several hours after the initial reaction had taken place. All enzyme reactions were buffered to pH 7.0. The steroid is stable at this pH. Higher values cause changes in the absorption spectrum. At pH 8.6, in Bicine buffer, the phenolic peak at 288 nm disappears within 15 min. Destruction of the steroid is even more rapid as the pH is raised. The peak at 288 nm is not affected at pH 7.5, but there is a slight increase in the trough at 256 nm. No change in the spectrum of 2-OH Ez was evident at pH 7.0 during the time course of these exper-

TABLE

II

RATEOFREACCIONOF&-HYDROXYESTRADIOLWITH ANDWITHOUTADDEDCATECHOL [2-Hydroxyestradiol] 04 2 x lo-” 2 x lo-’

[Catechol] (M) 2 x 10-S

&la (mol liter-’

s-i)

0.45 x 1o-6 8.1 x 10-e

Note. lOO(Rate in absence of catechol)/(rate in presence of catechol) = 5%. o Measured by absorbance difference at 256 and 206 nm.

194

JACOBSOHN

AND

mined whether tyrosinases from other sources have similar activities. Recently it was shown that 5,6-dihydroxyindole is a substrate for a mammalian tyrosinase which requires DOPA as a cofactor and produces melanochrome (5). The conditions for synthesizing 2-hydroxylated estrogens from their parent compounds require the presence of NADH or NADPH, a pH of 7.4, a temperature of 37”C, and mushroom tyrosinase at a level 3.5 times higher than used here (2). These conditions seem to preclude further oxidation of the catechol estrogen produced. The presence of a reduced dinucleotide, while necessary for the initial hydroxylation, may prevent the catecholase reaction. The latter reaction, as shown here, occurs under far milder conditions than the cresolase reaction but requires presence of catechol. Increases in the amount of catechol with fixed concentrations of 2-OH Ez (2 X 10V4 M) raises the rate of reaction (Figs. 3 and 6). The consumption rate of oxygen due to 2-OH Ez appears to level off at a concentration of catechol of 1 X 10e4 M. In the spectrophotometric assay, the activity of enzyme continues along a straight line up to the highest concentration of catechol tested, 2 X 10e4M. Whereas in the oxygenconsumption trials, the rates of oxygen loss in natoms was equal to nanomoles of steroid or catechol added within a limit of &lo% (data not shown), in the spectrophotometric determinations it was not possible to establish such a relationship. In the latter trials the cells were open to the atmosphere and oxygen was not limiting. In the polarographic tests, oxygen

iments. There was no measurable change in pH from beginning to the end of incubations. The concentration of oxygen is critical for enzyme activity. If the incubations were performed in a closed cell that had been purged with nitrogen for 12 min (Anaerobic cell accessory, Aminco), the rate of reaction decreased to l/4 of its former value (Table III). The (lower) small activity may be due to residual oxygen. The incubates, after reaction had taken place, were pooled and refrigerated until further use. When a sufficient amount had accumulated, they were brought to room temperature, and extracted and chromatographed as described under Materials and Methods. Estrone, estradiol, estriol, 2OH Ez, and an aliquot of the incubated sample extracts were plated on thin-layer chromatography plates and run in the system described. The eluting solvent was run up to 15 cm. The Rf values were estrone, 0.59; estradiol, 0.50; estriol, 0.17; 2-OH Ez, 0.41; and product extracted from incubates, 0.18. The latter showed extensive streaking beginning at the origin. DISCUSSION

Oxygen uptake data (Fig. 2) show that 2-OH Ez is unreactive at pH ‘7.0in the presence of mushroom tyrosinase unless catecho1 is added to the incubation medium. With catechol, the enzyme acts speedily in promoting oxidation of the estrogen. The tyrosinase-catalyzed oxygen uptake by an odihydroxy phenolic steroid is a novel effect of the enzyme and has not been described previously. It remains to be deter-

TABLE

[2-Hydroxyestradiol] 2 x lo-” 2 x lo-’

(M)

[Catechol] 1 x lo-’ 1 x lo-’

(M)

JACOBSOHN

III

Conditions Saturated with air Purged with Nz for 12 min

Note. lOO(Rate in absence of O&(rate in presence of 0,) = 25%. ’ Measured by absorbance difference at 256 and 206 nm.

Voo (mol liters-’ 2.19 x 1o-6 0.55 x 1o-6

9-l)

TYROSINASE

EFFECT

UPON

2-HYDROXYESTRADIOL

195

content was limited to the maximal sat- idized product no longer has the typical urating capacity of the incubation medium phenolic structure normally observed in at the start of the assay less the amount the 280 nm region. It is obscured by a large utilized by the oxidation of catechol. At increase in baseline so that only a shoulder the higher levels of catechol, oxygen dif- remains in this region. The unreacted stefusion may have prevented further in- roid dissolved in buffer was at a pH of 7.0, creases in maximal velocity of oxygen up- a value approximately midway between its assumed pK1 and pKz, by analogy with take by 2-OH Ez. The changes in the ultraviolet spectrum values obtained for 1,2-dihydroxybenzene noted above showed the disappearance of and many of its derivatives (8). At this pH the distinct phenolic peak after enzyme re- the concentration of the monodissociated action. The increase of absorbance at 256 ion is maximal (9). Since a hypsochromic nm and the constancy at 206 nm allowed shift is typical for the undissociated comfor quantitative measurements to be made pounds (8) and was observed here for the by dual-wavelength determinations of the steroid, one can assume that dissociatable difference between these two values, and groups are no longer present or are present the assumption that complete reaction has at reduced concentrations among the reaction products. On thin-layer chromatooccurred at the point where the light-absorption curve levels off. It had been noted grams, the migration rates of the products in the oxygen-consumption measurements are very slow and they remain partly at that product formation does not ceaseafter the origin. This is indicative of greater poconsumption of 1 natom oxygen/nmol ste- larity of the products or, more likely, an roid, but continues over an extended period increase in the molecular weight by interof time at a much reduced rate (Fig. 2). action of 2 or more molecules. The final This was observed also in the spectral de- products would be multimeric, analogous to melanin. Multimeric units based upon terminations (Fig. 5). The incubation products become visibly darker with time. the structure of a steroid estrogen have For this reason spectrophotometric mea- not been described before. Tyrosinase contains four copper atoms surements were preferably done by measuring transmittance at 100% calibration per molecule which are involved in the catrather than by direct absorbance mea- alytic activity (10). Azide forms strong surements. complexes with copper and produces a A decrease in the amount of available complicated inhibition pattern of tyrosioxygen causes a decrease in the rate of nase that has been interpreted in terms of reaction, determined spectrophotometriazide binding to both oxidized and reduced tally (Table III). The changes noted at 256 forms of the enzyme (11). Compounds posnm are, therefore, not due primarily to sessing aromatic rings and polar groups, binding of steroid to enzyme but are de- such as benzoic acid, also show inhibition pendent upon a primary effect of oxygen. of enzyme activity and act competitively The spectra of the steroid with and without (12). No clear view emerges on the manner enzyme show almost no difference (Fig. 4). by which catechol activates the enzyme toThe small changes which may be noted can ward 2-OH Ee. Catechol may itself, by rebe ascribed to a very low rate of reaction acting to yield oxidized products, free the in absence of added catechol; it was never enzyme of possible inhibitors present in more than 5% of the rate with catechol, the original source. A protein inhibitor Table II, and was frequently less. The small of human skin tyrosinase has been derate may be due to residual catechol or scribed (13). catechol-like substances contaminating the Other possibilities for catechol as a proenzyme or it may be due to actual enzyme moter of tyrosinase activity on 2-OH Ez activity unstimulated by catechol. can be considered. Catechol may function The products of the reaction with 2-OH as an activator, perhaps altering the conEz have not been identified. The initial, ox- formation of the enzyme to allow for in-

196

JACOBSOHN

AND

creased interaction between the steroid and the enzyme active site. It may promote increased binding of substrate and decreased binding of product. It has been suggested that DOPA promotes the hydroxylating activity of tyrosinase upon tyrosine (14) or the dehydrogenation of dihydroxyindole by the same enzyme (5). DOPA may function by eliciting a conformational change in tyrosinase (15). It is also possible that catecho1 and its oxidized product function as intermediate hydrogen carriers for the steroid to yield the corresponding semiquinone or quinone. If the semiquinone of the steroid were a product, then a subsequent nonenzymatic reaction may yield a di- or multimeric unit. Such compounds have been described for catechol (16). Alternatively, catechol may form a joint product with the oxidized dihydroxy steroid and thereby promote completion of the reaction by forming an adduct. REFERENCES 1. HERSEY, R. M., WILLIAMS, K. I. H., AND WEISZ, J. (1981) Endocrinology, 109,1912-1920. 2. JELLINCK, P. H., AND BROWN, B. J. (1971) steroid 17,133-140. 3. SEIJI, M., SHIMAO, K., BIRBEGK, M. S. C., AND FITZ-

JACOBSOHN

4. 5. 6. 7.

8. 9. 10. 11. 12. 13.

14.

15. 16.

PATRICK, T. B. (1963) Ann N y: Acd f&i. 109, 497-533. TOMITA, Y., HARIU, A., KATO, C., AND SEIJI, M. (1983) Arch Biochem Biophys 225, 75-85. KORNER, A., AND PAWALEK, J. (1982) Science 217, 1163-1165. LISBOA, B. P., AND DICZFALUSY, E. R. (1962) Acta Endmrinol 40, 60-81. ESTABROOK, R. W. (1967) in Methods in Enzymology (Estabrook, R. W., and Pullman, M. E., eds.), Vol. 10, pp. 41-47, Academic Press, New York. STAUDE, H., AND TEUPEL, M. (1953) Z. Ekldroc~ 61,181-187. SIJNKEL, J., AND STALJDE,H. (1969) Ber. Bunsenge-s Physik ChRm 73,203-209. BOUCHILL~UX, S., M~MAHILL, P., AND MASON, H. S. (1963) J. Biol Chem 238,1699-1707. HEALEY, D. F., AND STROTHKAMP, K. G. (1981) Arch, Biochem Biuphgs 211,86-91. DUCKWORTH, H. W., AND COLEMAN, J. E. (1970) J. Bid Chem 245,1613-1625. VIJAYAN, E., HUSAIN, I., R~AIAH,-A., AND MADAN, N. C. (1982) Arch. Biochem Biophys 217,738747. HEARING, V. J., EKEL, T. M., MONTAGUE, P. M., AND NICHOLSON, J. M. (1980) Biochim Biophys Acta 611,251-268. HEARING, V. J., AND EKEL, T. M. (1976) Biochem J. 157, 549-557. FORSYTH, W. G. C., AND Q~E~NEL, V. C. (1957) Biochim. Biophys. Actu 25,155-X0.