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J. Biol. Chem., Vol. 279, Issue 14, 13425-13434, April 2, 2004

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Stopped-flow Fluorescence Studies of Inhibitor Binding to Tyrosinase from Streptomyces antibioticus^*

Armand W. J. W. Tepper, Luigi Bubacco, and Gerard W. Canters¶

From the Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands and the Department of Biology, University of Padua, Via Trieste 75, 30121 Padua, Italy

Tyrosinase (Ty) is a type 3 copper protein involved in the rate-limiting step of melanin synthesis. It is shown that the endogenous Trp fluorescence of tyrosinase from Streptomyces antibioticus is remarkably sensitive to the redox state. The fluorescence emission intensity of the [(Cu(I) Cu(I)] reduced species is more than twice that of the oxygen-bound [Cu(II)--Cu(II)] form. The emission intensity of the oxidized [Cu(II)-OH^–-Cu(II)] protein (Ty_met) appears to be dependent on an acid-base equilibrium with a pK_a value of 4.5 ± 0.1. The binding of fluoride was studied under pseudo first-order conditions using stopped-flow fluorescence spectroscopy. The kinetic parameters k_on, K_d, and the fraction of fluorescence emission quenched upon fluoride binding show a similar pH dependence as above with an average pK_a value of 4.62 ± 0.05. Both observations are related to the dissociation of Cu₂-bridging hydroxide at low pH. It is further shown that Ty is rapidly inactivated at low pH and that halide protects the enzyme from this inactivation. All results support the hypothesis that halide displaces hydroxide as the Cu₂-bridging ligand in Ty_met. The relevance of the experimental findings for the catalytic cycle is discussed. The data are consistent with the data obtained from other techniques, validating the use of fluorescence quenching as a sensitive and effective tool in studying ligand binding and substrate conversion.

Received for publication, August 25, 2003 , and in revised form, December 19, 2003.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Leiden University, Einsteinweg 55, P. O. Box 9502, 2300 RA Leiden, The Netherlands. Tel.: 31-71-527-4256; Fax: 31-71-527-4349; E-mail: canters{at}chem.leidenuniv.nl.

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