Human Tyrosinase: Temperature-Dependent Kinetics of Oxidase Activity.

Kenneth L Young,David Eric Anderson,Yuri V Sergeev,Monika B Dolinska,Claudia Kassouf

doi:10.3390/ijms21030895

Abstract

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.

Highlights

Pigmentation is the result of a complex process by which the pigmentary biopolymer, melanin, is synthesized and packed into melanosomes of melanocytes
Various types of oculocutaneous albinism (OCA), a series of autosomal recessive disorders, are associated with reduced pigmentation in the skin, eyes, and hair due to malfunctioning proteins involved in melanogenesis, most notably, human tyrosinase where genetic mutations occur in its corresponding gene TYR
The recombinant Tyrtr was purified by a combination of immobilized metal affinity chromatography (IMAC) and size-exclusion chromatography (SEC) where purification steps were monitored by SDS-PAGE and Western blotting analyses

Summary

Introduction

Pigmentation is the result of a complex process by which the pigmentary biopolymer, melanin, is synthesized and packed into melanosomes of melanocytes. Oculocutaneous albinism type 1 (OCA1), the most wide-spread albinism, is caused by bi-allelic mutations in the TYR gene and occurs in approximately 1:40,000 people worldwide [1]. This type of albinism is divided clinically into two groups: the most severe type, OCA1A, with disrupted tyrosinase activity and melanin synthesis, or the less severe OCA1B, with residual tyrosinase activity in melanin production. More than 350 mutations were found in the TYR gene as noted in the HGMD Professional 2019.2 database (https://portal.biobase-international.com/hgmd/pro/) Many of these alterations change the production of melanin either by full or partial exclusion of tyrosinase activity

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Results

Conclusion