Structural Basis and Mechanism of the Inhibition of the Type-3 Copper Protein Tyrosinase from Streptomyces antibioticusby Halide Ions

Abstract

The inhibition of the type-3 copper enzyme tyrosinase by halide ions was studied by kinetic and paramagnetic 1 H NMR methods. All halides are inhibitors in the conversion of L-3,4-dihydroxyphenylalanine (L-DOPA) with apparent inhibition constants that follow the order I ؊ < F ؊ < < Cl ؊ < Br ؊ at pH 6.80. The results show that the inhibition arises from the interaction of halide with both the oxidized (affinity F ؊ > Cl ؊ > Br ؊ > > I ؊ ) and reduced (affinity I ؊ > Br ؊ > Cl ؊ > > F ؊ ) enzyme. The paramagnetic 1 H NMR of the oxidized enzyme complexed with the halides is consistent with a direct interaction of halide with the type-3 site and shows that the (Cu-His 3 ) 2 coordination occurs in all halide-bound species. It is surmised that halides bridge both of the copper ions in the active site. Fluoride and chloride are shown to bind only to the low pH form of oxidized tyrosinase, explaining the strong pH dependence of the inhibition by these ions. We further show that p-toluic acid and the bidentate transition state analogue, Kojic acid, displace chloride from the oxidized active site, whereas the monodentate substrate analogue, p-nitrophenol, forms a ternary complex with the enzyme and the chloride ion. On the basis of the experimental results, a model is formulated for the inhibitor action and for the reaction of diphenols with the oxidized enzyme.One of the unresolved questions in the enzymology of the type-3 copper-containing tyrosinases (EC 1.14.18.1) is the detailed molecular mechanism of both inhibitor action and substrate conversion. This report focuses on the mechanism of their inhibition by halides. Tyrosinases are monooxygenating enzymes catalyzing the ortho-hydroxylation of monophenols and the subsequent oxidation of the diphenolic products to the corresponding quinones. The reactions take place under concomitant reduction of molecular oxygen to water. The formed quinones are reactive precursors in the synthesis of melanin pigments. In fruits, vegetables, and mushrooms, Ty 1 is a key enzyme in the browning that occurs upon bruising or long-term storage. In mammals, Ty is responsible for skin pigmentation. Defects in the enzyme may lead to some forms of oculocutaneous albinism or vitiligo (1). Furthermore, the enzyme has been linked to Parkinson's and other neurodegenerative diseases (2-6). Consequently, the enzyme poses considerable interest from medical, agricultural, and industrial points of view.The current knowledge of Ty at the biological, mechanistic, and structural levels has recently been reviewed (7-10). Ty harbors a dinuclear so-called type-3 copper center, the occurrence of which has also been established in hemocyanins, which act as oxygen carriers in arthropods and mollusks, and the catechol oxidases, which oxidize o-diphenols to the corresponding quinones. The two closely spaced copper ions in the type-3 active site are coordinated each by 3 histidine residues through the N⑀ nitrogen atoms (9). Although the known type-3 centers are found to be similar both in structure and in their a...

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

“…halide only binds to the acidic form of the enzyme, resulting in stronger inhibition with decreasing pH. As a consequence of the instability of the enzyme at low pH and of the relatively long experimental times required, in the past we were able to estimate only an upper limit of 5.5 for the pK a of the process modulating the halide binding (26). In the same study it was also proposed that halides bridge the two copper ions in the active site.…”

“…pH-dependent Inactivation of Ty-From previous studies (26), it is known that the Ty from S. antibioticus is unstable at low pH. The closely related Ty from Streptomyces glaucescens (93% sequence identity with S. antibioticus Ty) is more stable in Tris⅐HCl buffer than in phosphate buffer, especially at the lower pH values (43).…”

“…Furthermore, large changes in the Ty met paramagnetic 1 H NMR spectrum are observed when inhibitors bind to the active site (25,26), allowing us to follow ligand binding and to obtain structural information on the complexes. For instance, it was shown that halides interact with both the oxidized and reduced Ty, where the affinity for halide ion and the mechanism of inhibition depend both on the nature of the halide ion and the oxidation state of the type 3 site (26) …”

“…It was a step forward recently when the oxidized Ty met derivative appeared amenable to paramagnetic NMR (8,25,26); the S ϭ 1 spin state of Ty met was found to be thermally accessible at room temperature, providing sufficient paramagnetism for the signals of the coordinating histidine protons to shift outside the diamagnetic envelope. The NMR studies demonstrated that also Ty contains a classical type 3 site in which, like in Hcs and COs, the two copper ions are coordinated by six histidine residues through their N⑀ atoms (8).…”

“…This report focuses on the fluorescence properties of the Ty from Streptomyces antibioticus as a function of pH and inhibitor binding. Fluoride was chosen (26) as a model compound to study the transient-state kinetics of its binding to the Ty met enzyme, as well as its pH dependence. Stopped-flow fluorescence spectroscopy was used to obtain insight into the interaction of fluoride with Ty, as well as to validate the use of fluorescence quenching in studying ligand binding kinetics.…”