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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...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2020
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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 |
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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 |
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