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Protein Stability and Functional Characterization of Intra-Melanosomal Domain of Human Recombinant Tyrosinase-Related Protein 1

Human Tyrosinase: Temperature-Dependent Kinetics of Oxidase Activity

National Eye Institute, National Institutes of Health, 31 Center Drive MSC 2510, Bethesda, MD 20892, USA
National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 6 Center Dr. MSC2775, Bethesda, MD 20892, USA
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2020, 21(3), 895;
Received: 4 November 2019 / Revised: 27 January 2020 / Accepted: 28 January 2020 / Published: 30 January 2020
(This article belongs to the Special Issue Advances in Biological Functions of Tyrosinase)
Human tyrosinase (Tyr) is involved in pigment biosynthesis, where mutations in its corresponding gene TYR have been linked to oculocutaneous albinism 1, an autosomal recessive disorder. Although the enzymatic capabilities of Tyr have been well-characterized, the thermodynamic driving forces underlying melanogenesis remain unknown. Here, we analyze protein binding using the diphenol oxidase behavior of Tyr and van ’t Hoff temperature-dependent analysis. Recombinant Tyr was expressed and purified using a combination of affinity and size-exclusion chromatography. Michaelis-Menten constants were measured spectrophotometrically from diphenol oxidase reactions of Tyr, using L-3,4-dihydroxyphenylalanine (L-DOPA) as a substrate, at temperatures: 25, 31, 37, and 43 °C. Under the same conditions, the Tyr structure and the L-DOPA binding activity were simulated using 3 ns molecular dynamics and docking. The thermal Michaelis-Menten kinetics data were subjected to the van ‘t Hoff analysis and fitted with the computational model. The temperature-dependent analysis suggests that the association of L-DOPA with Tyr is a spontaneous enthalpy-driven reaction, which becomes unfavorable at the final step of dopachrome formation. View Full-Text
Keywords: tyrosinase; protein purification; L-DOPA binding; enthalpy-driven association tyrosinase; protein purification; L-DOPA binding; enthalpy-driven association
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MDPI and ACS Style

Young, K.L., II; Kassouf, C.; Dolinska, M.B.; Anderson, D.E.; Sergeev, Y.V. Human Tyrosinase: Temperature-Dependent Kinetics of Oxidase Activity. Int. J. Mol. Sci. 2020, 21, 895.

AMA Style

Young KL II, Kassouf C, Dolinska MB, Anderson DE, Sergeev YV. Human Tyrosinase: Temperature-Dependent Kinetics of Oxidase Activity. International Journal of Molecular Sciences. 2020; 21(3):895.

Chicago/Turabian Style

Young, Kenneth L., II, Claudia Kassouf, Monika B. Dolinska, David E. Anderson, and Yuri V. Sergeev 2020. "Human Tyrosinase: Temperature-Dependent Kinetics of Oxidase Activity" International Journal of Molecular Sciences 21, no. 3: 895.

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