Abstract

Tyrosinase is a copper-binding enzyme involved in melanin biosynthesis. However, the detailed structure of human tyrosinase has not yet been solved, along with the identification of the key sites responsible for its catalytic activity. We used site-directed mutagenesis to identify the residues critical for the copper binding of human tyrosinase. Seven histidine mutants in the two copper-binding sites were generated, and catalytic activities were characterised. The tyrosine hydroxylase activities of the CuA site mutants were approximately 50% lower than those of the wild-type tyrosinase, while the dopa oxidation activities of the mutants were not significantly different from that of wild-type tyrosinase. By contrast, mutations at CuB significantly decreased both tyrosine hydroxylation and dopa oxidation activities, confirming that the catalytic sites for these two activities are at least partially distinct. These findings provide a useful resource for further structural determination and development of tyrosinase inhibitors in the cosmetic and pharmaceutical industries.

摘要

酪氨酸酶是参与黑色素生物合成的铜结合酶。然而，人酪氨酸酶的详细结构以及负责其催化活性的关键位点的鉴定尚未得到解决。我们使用定点诱变来鉴定对人酪氨酸酶的铜结合至关重要的残基。在两个铜结合位点产生了七个组氨酸突变体，并表征了催化活性。CuA位点突变体的酪氨酸羟化酶活性比野生型酪氨酸酶低约50％，而突变体的多巴氧化活性与野生型酪氨酸酶无明显差异。相比之下，CuB的突变会显着降低酪氨酸羟基化和多巴氧化活性，证实这两种活性的催化位点至少部分不同。这些发现为化妆品和制药工业中酪氨酸酶抑制剂的进一步结构测定和开发提供了有用的资源。