Characterization of Temperature-Dependent Kinetics of Oculocutaneous Albinism-Causing Mutant of Tyrosinase

Abstract

Purpose : Human tyrosinase (Tyr) is a glycoenzyme that catalyzes the first and rate-limiting steps 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 has only begun to be examined and remains to be delineated. Here, we analyze the temperature-dependent kinetics of wild type Tyr (WT) and R422Q mutant using Michaelis-Menten and van’t Hoff analysis.

Methods : Recombinant truncated human WT and R422Q mutant proteins (residues 19-469) were expressed in baculovirus and produced in whole insect Trichoplusia. Ni larvae. Proteins were purified by immobilized metal affinity chromatography followed by size-exclusion chromatography. Diphenol oxidase activities of WT and R422Q were measured using 0.05 mg/ml protein and L-DOPA as substrate at 28, 31, 37, and 43 degrees Celsius at physiological pH 7.4. Absorbance measurements of dopachrome formation were measured at 475 nm and converted to concentration. For Michaelis-Menten kinetics, absorbance measurements were performed at 0.09, 0.19, 0.38, 0.75, 1.5, 3, and 6 mM L-DOPA. Kinetic parameters of WT and R422Q catalyzed reactions were determined using nonlinear polynomial fit on OriginPro 7.5. The thermodynamic signature of dopachrome production was determined by measurement of Michaelis-Menten constant (K_m) followed by an analysis using the van’t Hoff equation.

Results : The Michaelis-Menten kinetics showed an increasing K_m (WT: R² = 0.87; R422Q: R² = 0.88) and V_max (WT: R² = 1.00; R422Q R² = 1.00) as the temperature increased. It also revealed that the V_max, k_cat, and k_cat/K_m are significantly higher for the WT compared to R422Q. The van’t Hoff analysis displayed a negative trendline for both the WT and R422Q. Subsequently, the ΔH (kJ/mol) and ΔS (kJ/mol*K) values were calculated (WT: ΔH = 21.25 ± 6.37, ΔS = 0.11 ± 0.02; R422Q: ΔH = 21.14 ± 6.09, ΔS = 0.11 ± 0.02), which resulted in a ΔG < 0 for both WT and R422Q.

Conclusions : Overall, the analysis of the temperature-dependent kinetics showed that R422Q is less active than WT at all temperatures examined here. Elucidating the thermodynamics and kinetics of both the WT and mutant Tyr is indispensable for understanding the molecular mechanism of OCA1 to discover drugs that may treat OCA1.

This is a 2021 ARVO Annual Meeting abstract.