Stopped-flow fluorescence studies of inhibitor binding to tyrosinase from Streptomyces antibioticus.
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)-O(2)(2-)-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 Cu(2)-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 Cu(2)-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.
Tepper
Armand W J W
AW
Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands.
Bubacco
Luigi
L
Canters
Gerard W
GW
D003300:Copper / Q000737:chemistry; D004789:Enzyme Activation; D005459:Fluorides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D014442:Monophenol Monooxygenase / Q000737:chemistry* / Q000378:metabolism*; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence; D013303:Streptomyces antibioticus / Q000201:enzymology*
D003300:Copper; D014442:Monophenol Monooxygenase; D005459:Fluorides