Wachamo Samuel A, Patel Milan H, Varghese Paul K, Dolinska Monika B, Sergeev Yuri V
National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Int J Mol Sci. 2021 Jul 21;22(15):7771. doi: 10.3390/ijms22157771.
Human tyrosinase (Tyr) is a glycoenzyme that catalyzes the first and rate-limiting step in melanin production, and its gene () 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 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.
人类酪氨酸酶（Tyr）是一种糖酶，它催化黑色素生成的第一步且是限速步骤，在许多1型眼皮肤白化病（OCA1）病例中其基因（）发生突变。OCA1患者中个体突变导致不同色素沉着表型的机制才刚刚开始研究，仍有待阐明。在此，我们使用米氏分析和范特霍夫分析来分析野生型Tyr（WT）以及两种OCA1B突变变体（R422Q和P406L）的温度依赖性动力学。重组截短的人类Tyr蛋白（第19 - 469位氨基酸残基）在全昆虫幼虫中产生。蛋白质通过亲和色谱和尺寸排阻色谱相结合的方法进行纯化。通过多巴色素吸收来测量二酚氧化酶蛋白活性和动力学参数的温度依赖性。在相同实验条件下，进行计算模拟以评估L - 多巴与Tyr的温度依赖性结合。我们的结果首次揭示，L - 多巴与R422Q和P406L结合后形成多巴色素是一个由焓和熵力支持的复杂反应。我们表明，与R422Q和P406L相比，WT具有更高的周转数。阐明OCA1B中Tyr突变变体的动力学和热力学有助于理解它们降低Tyr催化活性的机制，并为患者发现新的治疗方法。
