Mutational mapping of the catalytic activities of human tyrosinase.
Mutational mapping of the catalytic activities of human tyrosinase.
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DOI：
10.1016/s0021-9258(18)35895-2
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发表时间：
1992-11
期刊：
影响因子：
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通讯作者：
R. K. Tripathi;V. Hearing;K. Urabe;P. Aroca;R. Spritz
中科院分区：
文献类型：
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作者：
R. K. Tripathi;V. Hearing;K. Urabe;P. Aroca;R. Spritz
酪氨酸酶（EC 1.14.18.1）是一种含铜的金属糖蛋白，其催化黑色素生物合成途径中的几个步骤;酪氨酸羟基化为L-3，4-二羟基苯丙氨酸（多巴）以及随后多巴氧化为多巴醌。有人提出，酪氨酸酶还能够将黑素生成途径中的后期产物5，6-二羟基吲哚（DHI）氧化为吲哚-5，6-醌。酪氨酸酶的酶活性是缺乏典型的I型眼皮肤白化病（OCA）的患者，和超过50个不同的突变，现已确定在酪氨酸酶基因的患者。为了确定各种酪氨酸酶基因突变对酶催化活性的影响，我们对人酪氨酸酶cDNA进行了定点突变，在转染的HeLa细胞中瞬时表达突变的cDNA，并测定所得的酪氨酸羟化酶，多巴和DHI氧化酶活性的编码蛋白质，以及所得的黑色素产生。正常酪氨酸酶的酪氨酸羟化酶活性是热稳定的，而其多巴氧化酶和DHI氧化酶活性是温度敏感的。虽然测试的所有氨基酸取代通常平行地影响多巴氧化酶和DHI氧化酶的活性，但有几个对酪氨酸羟化酶活性产生明显不同的影响。总之，这些结果证实了哺乳动物酪氨酸酶的DHI氧化酶活性，并表明酪氨酸酶的多巴氧化酶和DHI氧化酶活性共享共同的催化位点，而酪氨酸羟化酶催化位点在酪氨酸酶多肽中至少部分不同。
Tyrosinase (EC 1.14.18.1) is a copper-containing metalloglycoprotein that catalyzes several steps in the melanin pigment biosynthetic pathway; the hydroxylation of tyrosine to L-3,4-dihydroxyphenylalanine (dopa) and the subsequent oxidation of dopa to dopaquinone. It has been proposed that tyrosinase is also able to oxidize 5,6-dihydroxyindole (DHI), a later product in the melanogenic pathway, to indole-5,6-quinone. Tyrosinase enzymatic activity is deficient in patients with classic type I oculocutaneous albinism (OCA), and more than 50 distinct mutations have now been identified in the tyrosinase genes of such patients. To determine the effects of the various tyrosinase gene mutations on the catalytic activities of the enzyme, we carried out site-directed mutagenesis of human tyrosinase cDNA, transiently expressed the mutant cDNAs in transfected HeLa cells, and assayed the resultant encoded proteins for tyrosine hydroxylase, dopa, and DHI oxidase activities, and resulting melanin production. The tyrosine hydroxylase activity of normal tyrosinase is thermostable, whereas its dopa oxidase and DHI oxidase activities are temperature-sensitive. Although all amino acid substitutions tested generally affected the dopa oxidase and DHI oxidase activities in parallel, several exerted distinctly different effects on the tyrosine hydroxylase activities. Together, these results confirm the DHI oxidase activity of mammalian tyrosinase and suggest that the dopa oxidase and DHI oxidase activities of tyrosinase share a common catalytic site, whereas the tyrosine hydroxylase catalytic site is at least partially distinct in the tyrosinase polypeptide.