Cargando…

Human Tyrosinase: Temperature-Dependent Kinetics of Oxidase Activity

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 underlyi...

Descripción completa

Detalles Bibliográficos
Autores principales: Young, Kenneth L., Kassouf, Claudia, Dolinska, Monika B., Anderson, David Eric, Sergeev, Yuri V.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7037427/
https://www.ncbi.nlm.nih.gov/pubmed/32019134
http://dx.doi.org/10.3390/ijms21030895
_version_ 1783500425531490304
author Young, Kenneth L.
Kassouf, Claudia
Dolinska, Monika B.
Anderson, David Eric
Sergeev, Yuri V.
author_facet Young, Kenneth L.
Kassouf, Claudia
Dolinska, Monika B.
Anderson, David Eric
Sergeev, Yuri V.
author_sort Young, Kenneth L.
collection PubMed
description 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.
format Online
Article
Text
id pubmed-7037427
institution National Center for Biotechnology Information
language English
publishDate 2020
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-70374272020-03-11 Human Tyrosinase: Temperature-Dependent Kinetics of Oxidase Activity Young, Kenneth L. Kassouf, Claudia Dolinska, Monika B. Anderson, David Eric Sergeev, Yuri V. Int J Mol Sci Article 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. MDPI 2020-01-30 /pmc/articles/PMC7037427/ /pubmed/32019134 http://dx.doi.org/10.3390/ijms21030895 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Young, Kenneth L.
Kassouf, Claudia
Dolinska, Monika B.
Anderson, David Eric
Sergeev, Yuri V.
Human Tyrosinase: Temperature-Dependent Kinetics of Oxidase Activity
title Human Tyrosinase: Temperature-Dependent Kinetics of Oxidase Activity
title_full Human Tyrosinase: Temperature-Dependent Kinetics of Oxidase Activity
title_fullStr Human Tyrosinase: Temperature-Dependent Kinetics of Oxidase Activity
title_full_unstemmed Human Tyrosinase: Temperature-Dependent Kinetics of Oxidase Activity
title_short Human Tyrosinase: Temperature-Dependent Kinetics of Oxidase Activity
title_sort human tyrosinase: temperature-dependent kinetics of oxidase activity
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7037427/
https://www.ncbi.nlm.nih.gov/pubmed/32019134
http://dx.doi.org/10.3390/ijms21030895
work_keys_str_mv AT youngkennethl humantyrosinasetemperaturedependentkineticsofoxidaseactivity
AT kassoufclaudia humantyrosinasetemperaturedependentkineticsofoxidaseactivity
AT dolinskamonikab humantyrosinasetemperaturedependentkineticsofoxidaseactivity
AT andersondavideric humantyrosinasetemperaturedependentkineticsofoxidaseactivity
AT sergeevyuriv humantyrosinasetemperaturedependentkineticsofoxidaseactivity