Abstract
Human tyrosinase (Tyr) is a glycoenzyme that catalyzes the first and rate-limiting step in melanin production, and its gene (TYR) is mutated in many cases of oculocutaneous albinism type 1 (OCA1). The mechanisms by which individual mutations contribute to the diverse pigmentation phenotype in patients with OCA1 have only began to be examined and remain to be delineated. Here, we analyze the temperature-dependent kinetics of wild-type Tyr (WT) and two OCA1B mutant variants (R422Q and P406L) using Michaelis–Menten and Van’t Hoff analyses. Recombinant truncated human Tyr proteins (residues 19–469) were produced in the whole insect Trichoplusia Ni larvae. Proteins were purified by a combination of affinity and size-exclusion chromatography. The temperature dependence of diphenol oxidase protein activities and kinetic parameters were measured by dopachrome absorption. Using the same experimental conditions, computational simulations were performed to assess the temperature-dependent association of L-DOPA and Tyr. Our results revealed, for the first time, that the association of L-DOPA with R422Q and P406L followed by dopachrome formation is a complex reaction supported by enthalpy and entropy forces. We show that the WT has a higher turnover number as compared with both R422Q and P406L. Elucidating the kinetics and thermodynamics of mutant variants of Tyr in OCA1B helps to understand the mechanisms by which they lower Tyr catalytic activity and to discover novel therapies for patients.
Highlights
Mutations in the tyrosinase gene (TYR) cause oculocutaneous albinism type 1 (OCA1), an autosomal recessive disorder characterized by a lack of melanin biosynthesis or reduced melanin pigment in the hair, skin, and eyes
We have shown that OCA1A related mutants (T373K and R77Q) and OCA1B related mutants (R402Q, R422W, R422Q, and P406L) influence protein folding, stability, and intrinsic activity
The apparent thermodynamic signature of dopachrome production was determined by the measurement of the Michaelis–Menten constant (Km ) followed by an analysis using decreased dopachrome production in mutant variants as compared with the wild-type Tyr (WT), sig ing decreased activity of mutant Tyr
Summary
Mutations in the tyrosinase gene (TYR) cause oculocutaneous albinism type 1 (OCA1), an autosomal recessive disorder characterized by a lack of melanin biosynthesis or reduced melanin pigment in the hair, skin, and eyes. OCA1A is the most severe type of OCA1, leading to a complete lack of melanin production due to loss of tyrosinase (Tyr) activity. The thermodynamics and kinetics of mutant variants of Tyr have yet to be elucidated to understand the mechanisms by which mutations contribute to the diverse pigmentation phenotype in OCA1 and to recover Tyr activity in patients. In vitro [6] studies have shown that there are temperature-sensitive mutant variants of Tyr that cause OCA1. We characterize the temperature-dependent kinetics and thermodynamic signatures of WT and two OCA1B mutants of Tyr, R422Q and P406L, using diphenol oxidase activities at 28, 31, 37, and 43 ◦ C. Elucidating the kinetics and thermodynamics of mutant variants of Tyr in OCA1B helps to understand the mechanisms by which they lower Tyr catalytic activity. In addition to in vitro studies, in vivo studies of similar nature coupled with investigation of other mutants would advance our understanding of mechanisms in OCA1B and lead to discoveries of novel therapies for OCA1B patients
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