Oxygen metabolism, essential to life in humans and other aerobic organisms, involves a variety of metalloenzymes. Many of the pathological consequences of their disruption remain poorly understood. Tyrosinase, found throughout the phylogenetic scale, catalyzes the first two steps in the biosynthesis of melanin: hydroxylation of phenols (a monooxygenase reaction) and the oxidation of catechols to quinones (an oxidase reaction). Melanin is a brown polymeric pigment which is responsible for skin, hair and eye pigmentation in humans and serves a variety of functions in other organisms. Both steps catalyzed by tyrosinase utilize molecular oxygen and represent two important types of oxygen-requiring reactions. Thus, investigation of the catalytic mechanism of tyrosinase will help elucidate the way metalloenzymes activate the oxygen molecule for various metabolic needs. Tyrosinase contains a binuclear copper site and the individual copper ions have been proposed to have specific roles in the monooxygenase and oxidase reactions. The two copper ions of the native binuclear site are indistinguishable, making it impossible to study their individual roles. A method of preparing unique mixed-metal derivatives containing one copper ion and either a cobalt or zinc ion in the binuclear site has been developed. The individual spectroscopic features of the two different metal ions will be exploited in order to investigate their interactions with substrates and other ligands These studies will allow testing and refinement of existing mechanistic proposals. The spectroscopic characteristics of the met apo derivative (in which only one copper of the binuclear site is present) and mixed-metal derivatives will also determine the influence of one metal ion on the coordination environment of the other and, in some cases, the magnetic interactions between the metal ions. Preparation of additional mixed-metal derivatives, using transition metals with different preferential coordination geometries, will allow assessment of the flexibility the metal binding sites and possible conformational changes in the binuclear site on the binding of substrates and other ligands.
| Conrad, J S; Dawso, S R; Hubbard, E R et al. (1994) Inhibitor binding to the binuclear active site of tyrosinase: temperature, pH, and solvent deuterium isotope effects. Biochemistry 33:5739-44 |
