Spectroscopy and Kineticsof Tyrosinase Catalyzed trans-Resveratrol Oxidation.

The spectroscopy and kinetics of the tyrosinase catalyzed trans-resveratrol oxidation were investigated by measuringboth UV–vis absorption spectra over the 200–500 nm rangeand Raman spectra over the 600–1800 cm–1region.Room temperature UV–vis absorption spectra, as a function oftime, showed the presence of two isosbestic points located at λ1= 270 nm and λ2= 345.5 nm delimiting twodifferent regions: the reactant region around 300 nm, where the absorptiondecreased with time, and the product region over the low wavelength(λ < 260 nm) and high wavelength (λ > 390 nm) wavelengthzone in which the absorption increased with time until, in both cases,constant values were achieved. A first-order kinetics was deducedwith a rate coefficient of k1= (0.10± 0.001) min–1, which turned out to be independentof substrate concentration over the 50–5 μM range; afeature that was rationalized by invoking the limiting case of theMichaelis–Menten scheme appropriate for substrate concentrationmuch lower than the respective Michaelis constant. The observationof the distinct resonance enhanced Raman lines, specifically thosepeaking at 830 cm–1, 753 cm–1,and 642 cm–1together with their time evolution,permitted us to gain insight into some crucial features and stepsof the catalytic reaction. Namely, that the formation of the so-called trans-resveratrol and tyrosinase SP complex withits O–O bridge plays a crucial role in the first steps of thisenzymatic reaction and that the hydroxylation of the ortho C–Hbond of the trans-resveratrol OH group occurs afterO–O bond cleavage in the tyrosinase activesite. The present study makes clear that a class of potential inhibitorsof tyrosinase can be found in compounds able to bind the two Cu (II)ions of the enzyme bidentate form. [ABSTRACT FROM AUTHOR]