Young, Kenneth L.Kassouf, ClaudiaDolinska, Monika B.Anderson, David EricSergeev, Yuri V.2020-03-022020-03-022020-01-30Young, 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.http://hdl.handle.net/10919/97090Human tyrosinase (Tyr) is involved in pigment biosynthesis, where mutations in its corresponding gene <i>TYR</i> 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 &rsquo;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 &deg;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 &lsquo;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.application/pdfenCreative Commons Attribution 4.0 Internationaltyrosinaseprotein purificationL-DOPA bindingenthalpy-driven associationArticle - Refereed2020-03-02https://doi.org/10.3390/ijms21030895