Spectroscopic Characterization of the Electronic Changes in the Active Site of Streptomyces antibioticus Tyrosinase upon Binding of Transition State Analogue Inhibitors

Abstract

The dinuclear copper enzyme tyrosinase (Ty) from genetically engineered Streptomyces antibioticus has been investigated in its paramagnetic half-met form [Cu(I)-Cu(II)]. The cw EPR, pulsed EPR, and hyperfine sublevel correlation spectroscopy (HYSCORE) experiments on the half-met-Ty and on its complexes with three different types of competitive inhibitor are reported. The first type includes p-nitrophenol, a very poor substrate for the monooxygenase activity of Ty. The second type comprises hydroxyquinones, such as kojic acid and L-mimosine, and the third type of inhibitor is represented by toluic acid. The electronic and structural differences of the half-met-Ty form induced at the cupric site by the different inhibitors have been determined. Probes of structural effects are the hyperfine coupling constants of the non coordinating N␦ histidyl nitrogens. By using the available crystal structures of hemocyanin as a template in combination with the spectroscopic results, a structural model for the active site of half-met-Ty is obtained and a model for the binding modes of both mono-and diphenols could be proposed.

Investigation of Binding‐Site Homology between Mushroom and Bacterial Tyrosinases by Using Aurones as Effectors

“…For TyM1, we used a commercially available A. bisporus Ty, purified by Q‐sepharose FF chromatography according to a known procedure 24. The S. antibioticus TyB3 was produced in the strain S. antibioticus pIJ703 and purified according to a published procedure 21. For both enzymes, we determined the Michaelis–Menten kinetic parameters for L ‐DOPA oxidation by plotting absorbance at 475 nm against time (formation of DOPAchrome).…”

“…For this, we compared the activities of A. bisporus tyrosinase (TyM1) and Streptomyces antibioticus tyrosinase (TyB3). The choice of TyB3 was motivated by our expertise in its expression and purification 21. In addition, TyB2 and TyB3 belong to the same Streptomyces genus and share more than 82 % identity (Table S1).…”

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

“…On this basis a model for diphenol coordination was proposed whereby one of the phenolic oxygens bridges the two copper ions by displacing the bridging hydroxide that is present in native Ty met (26). The displacement of an equatorial water ligand by bidentate ligands has also been shown to occur for half-oxidized Ty (Ty half-met ) by means of pulsed EPR spectroscopies (60,61). For oxidation of a diphenolic substrate to proceed, the diphenol must lose two protons in the process.…”

Binding of 2-Hydroxypyridine-N-oxide on Dicopper(II) Centers: Insights into Tyrosinase Inhibition Mechanism by Transition-State Analogs

“…These results, which are of biological relevance in the context of Ty mechanism, has to be placed in the perspective of the work of Woolery et al with the L-mimosine as adduct of Neurospora crassa Ty 24 and the more recent one of Bubacco et al with kojic acid as adduct of Streptomyces antibioticus Ty. 25 In this later study, on the basis of an X-ray absorption analysis, 25 ESEEM, 26 and HYSCORE, 27 kojic acid was proposed to be a bidentate ligand to CuB of Ty with one O-atom axially coordinated and the second probably bridging the two copper ions in a η 2 :η 1 bidentate mode. These finding are of great interest in relation with new Ty inhibitor design.…”