Biochem. J. (1992) 282, 915-918 (Printed in Great Britain)

915

Stabilization of the oxy form of tyrosinase by amino acid substitution

a

single conservative

Martin P. JACKMAN,* Marcel HUBER, Alex HAJNAL and Konrad LERCHt Biochemisches Institut der Universitat Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland

Asp-208 of Streptomyces glaucescens tyrosinase (an invariant residue in the CuB-binding region of tyrosinases and haemocyanins) was conservatively substituted by glutamic acid. Although having little effect on spectroscopic or kinetic properties of the enzyme, the mutation greatly decreased the lability of Cu-bound 02 A rationalization for these results is given, based on the crystal structure of Panuliris interruptus haemocyanin in the conserved CuB-binding region.

INTRODUCTION The mono-oxygenase tyrosinase (EC 1.14.18.1) (Ty) is responsible for the formation of melanin and related pigments by catalysing the o-hydroxylation of monophenols and the twoelectron oxidation of o-diphenols to o-quinones [1,2]. The enzyme is ubiquitously distributed, from Gram-positive bacteria to man

[3].

Physiochemical studies have shown that the enzyme's active site contains a coupled binuclear copper complex possessing remarkably similar spectral properties to those of haemocyanins (Hc), the 02-transport protein of molluscs and arthropods [4-7]. Thus the naturally occurring oxy (Cu",2022-), met (Cu"12) and deoxy (Cu'2) forms as well as the carbonyl and azide derivatives of both copper proteins have been described [3,8]. Three-dimensional structural information is at present restricted to Hc from the arthropod Panulirus interruptus. The refined structure of deoxy-Hc [9,10] reveals a pseudo-two-fold active-site symmetry with each of the copper atoms, designated CuA and CuB' ligated by three histidine residues. Comparison of many Ty and Hc primary structures [10-12] has led to the proposal of a common CuB-binding region for these proteins, encompassing the three putative histidine ligands and a highly conserved sequence of 19 residues that constitutes a-helix 2.6 (residues 377-395) in the P. interruptus Hc crystal structure [10]. In contrast, the CuA-binding sites of Ty and Hc appear to be rather variable [11]. These observations have been reinforced by site-directed mutagenesis experiments on Streptomyces glaucescens Ty [13,14]. Replacement of the histidine residues corresponding to the CUB ligands of P. interruptus Hc by asparagine or glutamine resulted in loss of active-site copper and drastically decreased activity in the mutant enzymes, implying their involvement as Cu ligands. Mutation of further targeted histidine residues has allowed the tentative assignment of a novel CuA-binding site in this enzyme [13,14]. Here we report the conservative substitution of Asp-208 of S. glaucescens Ty by glutamic acid (mutant D208E). This invariant aspartic acid residue lies within the highly conserved region, preceding CuB-ligand His-215 by seven residues. The spectroscopic and kinetic properties of native and mutant enzymes are compared, and the results cautiously interpreted with reference to the crystal structure of P. interruptus Hc.

MATERIALS AND METHODS Mutagenesis and expression of the tyrosine gene Site-directed mutangenesis was performed by the Kunkel method [15] with the use of the uracil-containing 480 bp PstI-BamHI fragment of the S. glaucescens Ty gene [16] cloned into M13mp19, and the anti-sense mutagenic primer 5'-CACCGGCTCGTTGGG-3'. Details of mutagenesis and amplification have been published previously [13,14]. The identity of the mutation was confirmed by chain-termination DNA sequencing [17].

Tyrosinase expression and purification The 2.2 kbp KpnI fragment containing the complete Ty gene was inserted into Streptomyces vector pMEA4 [18] and expressed in S. glaucescens by following literature procedures [13]. Mutant and native enzymes were purified by using a published method [14] in which CuCl2 (10 tM) was present throughout chromatography in order to suppress loss of copper from the active site. After the purification, extraneous Cu(II) was removed by passing the protein sample through a Sephadex G-25 column equilibrated in copper-free 10 mM-Tris/HCl buffer, pH 8.6. The resulting enzymes were purified to homogeneity, judged from SDS/12.5 %-PAGE gels [19] stained with Coomassie Blue G250. Samples were used immediately for further experiments, since storage results in slow pigmentation and inactivation. Protein concentrations were calculated by using the absorption coefficient 62808.21 x 104 M-l cm-l [14] determined for native Ty.

Spectroscopic and kinetic measurements All spectroscopic measurements were made in 10 mM-Tris/HCl buffer, pH 8.6. Copper concentrations were determined with the use of the Cu(I)-chelator bathocuproinedisulphonic acid [20]. Measurements were confined to the range 0.4-10 ,ug of Cu/ml, within the linear region of a calibration curve constructed with standard dilutions of CuCl2 (Titrisol; Merck). Maximal oxyenzyme formation was achieved by adding hydroxylamine in 3fold molar excess over copper. Absorption spectra were measured at 10 °C on a Kontron Uvicon 860 spectrometer. C.d. spectra of the oxy-enzymes and luminescence spectra of the carbonyl derivatives were obtained as previously described [13,14]. In the

Abbreviations used: Ty, tyrosinase; Hc, haemocyanin. * To whom correspondence should be sent, at present address: Friedrich Miescher Institut, P.O. Box 2543, CH-4002 Basel, Switzerland. t Present address: Givaudan Forschungsgesellschaft, Uberlandstrasse 138, CH-8600 Dubendorf, Switzerland.

Vol. 282

M. P. Jackman and others

916

oxy-enzyme stability experiments, bovine liver catalase (Boehringer) was added after 45 min to a final concentration of 40 nM, in order to scavenge for free peroxide ions. Spectrophotometric assays for detection of Ty during purification were performed at 30 °C in 0.1 M-sodium phosphate buffer, pH 6.0, with L-3,4-dihydroxyphenylalanine (7 mM) as substrate. One unit is defined as 1.8 A units/min at 475 nm [21]. For kinetic studies presented in Table 2, 02-consumption assays were performed at 30 °C as previously described [22]. Organic substrate concentrations were in the range 0.2-20 mm. Determination of apparent 02 affinity was performed with L-3,4dihydroxyphenylalanine (6 mM) as substrate. Data were fitted by Eadie-Hofstee plots [23], yielding correlation coefficients of between -0.79 and -0.95.

RESULTS AND DISCUSSION Choice of mutation Amino acid sequences of Ty and arthropodan and molluscan Hc corresponding to a-helix 2.6 in the Cue-binding region of P. interruptus Hc [10] were aligned. The representative alignment shown in Fig. 1 indicates the five residues that are invariant in all sequences yet published. In S. glaucescens Ty these residues are Asp-208, Pro-209, Phe-21 1, His-215 and Asp-219. The mutation D208E was chosen after referring to the P. interruptus Hc crystal structure. The equivalent residue (Asp-377) was found to be hydrogen-bonded to an arginine residue (Arg-271). If the invariance of this aspartic acid residue reflects an similar function in S. glaucescens Ty, its conservative mutation to glutamic acid might be expected to modify but not destroy the enzyme activity. Expression screening of D208E yielded black colonies on mel-

anin-indicator plates [18], indicating that the mutant did indeed have an active phenotype. Spectroscopic properties The copper contents of purified native Ty and D208E (Table 1) are in satisfactory agreement with the value of 2.0 mol/mol of enzyme expected for a binuclear active site. In the oxy forms of Ty and Hc, 02 is bound as a ,t-1,2-peroxo complex, yielding the characteristic O2- -+ Cu(II)2 charge-transfer spectrum with an intense peak at approx. 345 nm [6-8]. Oxy-Hc and Oxy-Ty are in equilibrium with both the met and deoxy forms [27], as illustrated in Scheme 1. To prepare the oxy-enzyme, hydroxylamine was added in slight excess over copper in order to reduce met-Ty to the deoxy form, which binds molecular 02 under aerobic conditions [28]. Hydroxylamine was used in preference to direct reaction of met-Ty with H202, since the former was found to yield the oxy-enzyme quantitatively [28]. Increasing the partial pressure of 02 in the mutant enzyme solution did not further enhance the absorption at 345 nm, confirming that oxyenzyme formation had reached completion. The spectral peak positions closely matched those of native Ty (Table 1), indicating no major perturbation of the D208E active site. Moreover, the intensity of the 345 nm peak was decreased by- only 30% (u.v.-visible spectrum) or 41 % (c.d. spectrum) in the mutant enzyme. Larger changes in c.d. peak intensity at longer wavelengths (Table 1) may, however, indicate some perturbation of the copper d-d transitions [30]. The labilities of the oxy-enzymes were monitored as a function of time (Fig. 2). The results were reproduced on two separate occasions, each with independently prepared samples of both native and mutant Ty. The decay in A345 for native Ty was linear

E Ty

Bacterial (S. glaucescens) Mammalian (H. sapiens) Ascomycete (N. crassa)

(208) - [ P V F W L H H A Y Y D R'J W1A ErFWQ (369) - D P I F L L H H A F V D S I FID QIWIL (299) - D P L F L L H H Y N Y D RIL WiS IIWIQ

(199) -D P V F F L H H A N T D Arthropod (f! interruJptuls) ( 377 ) - D P S IFIF R L H* K Y M

Hc Mollusc(H.pomatia)

RIL

WIA IIWIQ JKKLj T

Fig. 1. Alignment of primary structures corresponding to the region of ao-helix 2.6 in the P. interruptus Hc structure Invariant or highly conserved residues are shown in continuous or broken boxes respectively. The mutation described is indicated with an arrow and the site of Cu-binding by *. Source of sequences: Ty: S. glaucescens [16], Homo sapiens [24], N. crassa [25]; Hc: H. pomatia subunit d [26], P. interruptus [27]. Table 1. Comparison of the properties of purified native and mutant tyrosinases

Conditions are as described in the Materials and methods section. Values for the native enzyme are taken from refs. [13] and [14]. Abbreviation: L-dopa, L-3,4-dihydroxyphenylalanine. Native enzyme

Cu content (mol/mol of enzyme) Oxy-enzyme u.v.-visible absorption Amax (nm)[10-3 Xe (M cm-1)] Oxy-enzyme chiroptical properties Amax (nm) [I0O- x 0 (degree -cm2 -dmol -)] CO derivative, luminescence emission Amax (nm) (relative intensity) L-Dopa oxidase activity (units/mg)

Mutant D208E

1.8 +0.1 345 (12.7)

1.7+0.1 344 (8.9)

345 (-32.5) 470 (+2.1) 575 (- 1.7) 740 (+ 5.0) 538 (1.00)

344 (-19.1) 470 (+0.5) 575 (-2.5) 740 (+0.5) 538 (0.92)

2500+ 50

1100±50

1992

Stabilization of the oxy form of tyrosinase

917

Cu(II)202 202-

02

2e

Cu(II)2 '====~CU(I)2

Met

Deoxy Scheme 1. Equilibrium between met, oxy and deoxy forms of Ty

Arg-271

Fig. 3. Active-site structure of P. interrruptus Hc The Figure shows a view of the a-helices bearing the Cu ligands. The hydrogen-bonding interactions between Arg-271 and Asp-377 of ahelix 2.6 are indicated.

0

spectrum when excited at 275 nm. It has been proposed that CO binding occurs at CuA [9,14,33]. Interestingly, the carbonyl luminescence spectrum of D208E is almost identical with that of native Ty (Table 1), suggesting that the CuA environment is not perturbed by the effect of mutation.

20

40 Time (min) Fig. 2. Comparison of oxy-enzyme labilities at 10 °C 0, Native Ty (9.1 ,tM); 0, mutant D208E (9.8/,M). The time of catalase addition is indicated by an arrow.

point

under the conditions described in the Materials and methods section, with apparent rate constants of 4.4 x 10-4 min-' in the absence and 1.07 x 10-3 min-' in the presence of 40 nM-catalase. The faster decay in the presence of catalase demonstrates that an equilibrium between free and bound peroxide had been established. As expected, catalase increased the rate of decay of oxy-enzyme by removing 022, thus shifting the equilibrium in Scheme 1 in favour of met-Ty. For mutant D208E, however, the A345 was constant over the whole time course of the experiment, both before and after the addition of catalase. Setting an upper limit of I x 10-5 min-' for the apparent decay rate constant, this represents at least a 40-fold decrease in lability of enzyme-bound peroxide. In contrast with the Cu-bridged nature of bound peroxide, Ty and Hc each bind a single CO molecule in a terminal nonbridging manner [31,32], giving rise to an unusual fluorescence

Kinetic properties Kinetic rate constants for reaction of native and mutant Ty with monophenol and diphenol substrates and 02 are presented in Table 2. The mutation clearly did not affect the Km of organic substrates and caused only a nominal decrease in catalytic rate constant of 2-3-fold for both o-hydroxylation and two-electron oxidation. A similar effect was observed for the reaction with 02 with L-3,4-dihydroxyphenylalanine as organic substrate. It should be noted that the equilibrium described in this case is for binding of molecular 02 and not of peroxide (see Scheme 1), perhaps explaining why a larger effect is not observed. In summary, the conservative replacement D208E greatly enhances the stability of the oxy-enzyme with respect to the met form and free peroxide, while causing only small perturbations in the spectroscopic and kinetic properties of the enzyme. A tentative explanation for these results can be found by examining the crystal structure of P. interruptus Hc [9,10]. a-Helix 2.6, containing Cu ligand His-384, appears to be 'tethered' at its base

Table 2. Kinetic parameters for native tyrosinase and mutant D208E with a range of substrates

Conditions are as described in the Materials and methods section. Abbreviations: L-Dopa, dihydroxyphenylpropionic acid; 4-Hpa, 4-hydroxyphenylpropionic acid.

Mutant D208E

Native enzyme Substrate

L-Dopa

3,4-Dhpa L-Tyrosine 4-Hpa

02 Vol. 282

kcat (s1') 1260 1470 82 193 660

Km (mM)

5.8 1.3 2.4 0.4

0.084

L-3,4-dihydroxyphenylalanine; 3,4-Dhpa, 3,4-

105 x kcat/Km (M' .

2.2 12 0.35 4.8 79

l)

kcat.

Km

Km (M-1 . s-1)

1 x

(s1')

(mM)

680 930 22 100 340

6.3

1.1

1.3 2.0

7.2 0.11 2.0 48

0.5 0.071

918 by the salt bridge formed between Asp-377 and Arg-271 (Fig. 3). The sequence forming a-helix 2.6 is highly conserved between Hc and Ty (Fig. 1), suggesting that a similar tethering mechanism may operate in S. glaucescens Ty. Extending the length of the tether by inserting a -CH2- group (mutant D208E) could cause strain to be transmitted through the rigid a-helix to Cu ligand His-215, altering the CUB geometry without affecting the CUA environment. Such an effect might be expected to influence the binding of bridged ligands (e.g. peroxide) more strongly than that of non-bridged ligands such as CO, consistent with the spectroscopic results observed here. However, this proposal should be treated with caution: the region of P. interruptus Hc sequence containing Arg-271 is not conserved in Ty [11], and thus a putative hydrogen-bonding partner for Asp-208 of Ty cannot readily be identified. A conclusive interpretation must await structural data for a closely related Ty molecule. We gratefully acknowledge the Royal Society of Great Britain and the Roche Research Foundation for financial support (M. P. J.), and Dr. W. Hol for providing the crystal-structure co-ordinates of P. interruptus Hc.

REFERENCES 1. Mason, H. S. (1965) Annu. Rev. Biochem. 34, 594-634 2. Lerch, K. (1988) in Advances in Pigment Cell Research (Bagnara, J. T., ed.), pp. 85-89, Alan R. Liss, New York 3. Lerch, K. (1981) Met. Ions Biol. Syst. 13, 143-186 4. Schoot Uiterkamp, A. J. M. & Mason, H. S. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 993-996 5. Lerch, K. (1983) Mol. Cell. Biochem. 52, 125-138 6. Eickman, N. C., Solomon, E. I., Larabee, J. A., Spiro, T. G. & Lerch, K. (1978) J. Am. Chem. Soc. 100, 6529-6531 7. Himmelwright, R. S., Eickman, N. C., LuBien, C. D., Lerch, K. & Solomon, E. I. (1980) J. Am. Chem. Soc. 102, 7339-7344 8. Solomon, E. I. (1981) in Copper Proteins (Spiro, T. G., ed.), pp. 42-207, John Wiley and Sons, New York 9. Volbeda, A. & Hol, W. G. J. (1989) J. Mol. Biol. 206, 249-279

M. P. Jackman and others 10. Volbeda, A. & Hol, W. G. J. (1989) J. Mol. Biol. 206, 531-546 11. Lerch, K., Huber, M., Schneider, H., Drexel, R. & Linzen, B. (1986) J. Inorg. Biochem. 26, 213-217 12. Muller, G., Ruppert, S., Schmid, E. & Schutz, G. (1988) EMBO J. 7, 2723-2730 13. Huber, M. & Lerch, K. (1988) Biochemistry 27, 5610-5615 14. Jackman, M. P., Hajnal, A. & Lerch, K. (1991) Biochem. J. 274, 707-713 15. Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 488-492 16. Huber, M., Hintermann, G. & Lerch, K. (1985) Biochemistry 24, 6038-6044 17. Sanger, F., Nicklen, S. & Coulsen, A. R. (1979) Proc. Natl. Acad. Sci. U.S.A. 74, 5463-5468 18. Hintermann, G., Zatchej, M. & Hutter, R. (1985) Mol. Gen. Genet. 200, 422-432 19. Laemmli, U. K. (1970) Nature (London) 227, 680-685 20. Skotland, T. & Ljones, T. (1979) Eur. J. Biochem. 94, 145-151 21. Fling, M., Horowitz, N. H. & Heinemann, S. F. (1963) J. Biol. Chem. 238, 2045-2053 22. Toussaint, 0. & Lerch, K. (1987) Biochemistry 26, 8567-8571 23. Hofstee, B. H. J. (1959) Nature (London) 184, 1296-1298 24. Kwon, B. S., Haq, A. K., Pomerantz, S. H. & Halaban, R. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7473-7477 25. Lerch, K. (1978) Proc. NatI. Acad. Sci. U.S.A. 75, 3635-3639 26. Drexel, R., Schneider, H. J., Sigmund, S., Linzen, B., Gielens, C., Lontie, R., Preaux, G., Lottspeich, F. & Henschen, A. (1986) in Invertebrate Oxygen Carriers (Linzen, B., ed.), pp. 255-258, Springer-Verlag, New York 27. Linzen, B., Soeter, N. M., Riggs, A. F., Schneider, H.-J., Schartau, W., Moore, M. D., Yotota, E. A., Behrens, P. Q., Nakashima, I., Tagaki, T., Nemoto, T., Vereijken, J. M., Bak, H. J., Bientema, J. J., Volbeda, A., Gaykema, W. P. J. & Hol, W. G. J. (1985) Science 229, 519-524 28. Deinum, J., Lerch, K. & Reinhammer, B. (1976) FEBS Lett. 69, 161-164 29. Reference deleted 30. Himmelwright, R. S., Eickman, N. C., LuBien, C. D., Lerch, K. & Solomon, E. I. (1980) J. Am. Chem. Soc. 102, 7339-7344 31. Yen Fager, L. & Alben, J. 0. (1972) Biochemistry 11, 4786-4792 32. Kuiper, H. A., Lerch, K., Brunori, M. & Finazzi-Agr6, A. (1980) FEBS Lett. 111, 232-234 33. Sorrell, T. H., Beltramini, M. & Lerch, K. (1988) J. Biol. Chem. 263, 9576-9577

Received 6 August 199 1/1 November 1991; accepted 13 November 1991

1992