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Kenneth Lee Young II, Claudia Kassouf, Monika Dolinska, David Anderson, Yuri V. Sergeev; Title: Human Tyrosinase: Temperature-dependent Estimation of Gibbs Energies of Oxidase Activity. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5684. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Human Tyrosinase (Tyr) is involved in pigment biosynthesis–where mutations in its corresponding gene TYR have been linked to oculocutaneous albinism (OCA), a series of autosomal recessive disorders. Although the enzymatic capabilities of Tyr have been well characterized, the thermodynamic driving forces underlying melanogenesis have remained unknown. Previously, we have purified the human recombinant intra-melanosomal domain of Tyr denoted as Tyrtr. Here, we further characterize protein stability and elucidate the thermodynamic parameters behind the diphenol oxidase behavior of Tyrtr.
Recombinant Tyrtr was individually expressed in wholeTrichoplusia ni(T. ni) larvae and purified using immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC). Protein identity was confirmed using specific antibodies and mass spectrometry. Isothermal titration calorimetry (ITC) was used to corroborate and correlate Michaelis-Menten data obtained from L-DOPA oxidase activity of Tyrtr via UV spectroscopy. Gibbs free energies of product dissociation were calculated by measuring diphenol oxidase reactions of L-DOPA at four static temperatures: 25, 31, 37, 43°C spectroscopically at 475 nm.
Eluted as a single peak from SEC, Tyrtr exhibited a monomeric molecular weight of 57kDa. ITC was done in tandem with UV spectroscopic analysis of dopachrome formation yielding: Km7.03 (µM), Vmax0.01250 (µM/s), and Kcat6.66*10-6(s-1) at 37°C. Diphenol oxidase activity of L-DOPA was measured at different temperatures providing the following Km values: 0.357 mM (25°C), 0.456 mM (31°C), 0.575 mM (37°C), 0.475 mM (43°C). Generation of a Van’t-Hoff model was used to calculate Gibbs free energy, Enthalpy, and Entropy of dissociation, whereΔGd=30.53 kJ*K-1*mol-1, ΔHd =30.69 kJ* mol-1, and ΔSd =0.0005 kJ*K-1*mol-1.
From ITC, the diphenol oxidation overall is exothermic. From UV spectroscopy, the dissociation of product from Tyr is non-spontaneous and endothermic, meaning the binding is thermodynamically favorable. By understanding the energetics associated with Tyr binding, we can further comprehend how the active site functions, which is pivotal for the search of suitable activators and inhibitors of mutant variants. Moreover, examining the thermodynamics and kinetics of mutant Tyr remains vital in understanding the molecular mechanism of oculocutaneous albinism.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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