Document Type: Article
Department of Biology, Faculty of Basic Sciences, Islamic Azad University Science and Research Branch, Tehran, Iran
Cellular and Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, Iran
Background: Mushroom Tyrosinase a potent candidate in clinical studies known as polyphenol oxidase, is a metaloenzyme from the oxidase superfamily widely distributed from lower to higher life forms. It plays a crucial role in sclerotization of exoskeleton in insects, also responsible for skin pigmentation in mammalians.
Objective: In this study, after reconstitution of MT by some metal ions, the activity and structure of native, apo and reconstituted enzymes were investigated.
Materials and Methods: Kinetic of reconstituted tyrosinase carried out in catecholase reaction by depletion of caffeic acid. Tertiary and secondary structure of apo, native and metal reconstituted Tyrosinase obtained with fluorescence and circular dichroism techniques respectively. Reconstitution confirmed by Atomic Absorption Spectroscopy.
Results: kinetic assessment showed higher activity of MT reconstituted by Cu2+ and Ni2+ in comparison with native, Zn2+ and Co2+ reconstituted enzyme. The tertiary structure of enzyme by fluorescence technique indicated more stability of Ni2+ reconstituted MT and the apo form showed the lowest tertiary structure. Circular dichroism study showed that Ni2+ reconstituted MT form has more regular secondary structure and it caused higher stability of the enzyme. The molar ratio values from atomic absorption indicate that Ni2+ and Cu2+ have got the most binding to the apoenzyme.
Conclusions: It has been shown that Ni2+, Zn2+ and Co2+ can replace Cu2+ in tyrosinase, indicating that the histidines at the active site of the tyrosinase family enzymes can reconstitute with this metals, but, the most stabilization and well-structured enzyme was observed in the apotyrosinase reconstituted by Ni+2.