Sugumaran M, Bolton J L
Department of Biology, University of Massachusetts at Boston 02125, USA.
Arch Biochem Biophys. 1998 May 15;353(2):207-12. doi: 10.1006/abbi.1998.0653.
Tyrosinase, which is known to possess both monophenol monooxygenase activity (EC 1.14.18.1, tyrosine, 3,4-dihydroxyphenylalanine:oxygen oxidoreductase) and o-diphenoloxidase activity (EC 1.10.3.1, o-diphenol:oxygen oxidoreductase), has been shown to exhibit other related activities. Recently, a new reaction, viz., oxidative conversion of 2,6-dimethoxyallyl phenol to its quinone methide, catalyzed by commercial preparations of mushroom tyrosinase was reported (E. S. Krol, and J. L. Bolton, 1997, Chem. Biol. Interact. 104, 11-27). Since the reaction involves an unusual 1,6-oxidation rather than the conventional 1,4-oxidation, we reexamined this reaction more carefully. The o-diphenoloxidase activity and the dimethoxyallyl phenol oxidase activity of mushroom tyrosinase preparations exhibited different mobilities on size-exclusion chromatography on a Sephacryl S-200 column. A similar behavior was also witnessed on preparative isoelectric focusing in a rotofor cell. Different preparations of mushroom tyrosinase possessed varying ratios of these two activities, further confirming that they are due to two different enzymes. Native polyacrylamide gel electrophoresis followed by activity staining of the gel revealed different mobilities for these two activities. The protein band exhibiting dimethoxyallyl phenol oxidase activity could also be stained by syringaldazine, a well-known substrate for laccase (EC 1.10.3.2, p-diphenol:oxygen oxidoreductase). Two insect phenoloxidases, which are known for their wide substrate specificity, failed to oxidize dimethoxyallyl phenol to any detectable extent, thereby confirming that typical o-diphenoloxidases lack the ability to oxidize dimethoxyallyl phenol. On the other hand, laccase, which is known to convert syringaldazine to its quinone methide derivative, readily produced the quinone methide from dimethoxyallyl phenol. It is therefore concluded that laccase, which is present as a contaminant in the commercial preparations of mushroom tyrosinase--and not tyrosinase (o-diphenoloxidase)--is the enzyme responsible for catalyzing the new conversion of dimethoxyallyl phenol to its corresponding quinone methide.
酪氨酸酶已知具有单酚单加氧酶活性（EC 1.14.18.1，酪氨酸，3,4 - 二羟基苯丙氨酸：氧氧化还原酶）和邻二酚氧化酶活性（EC 1.10.3.1，邻二酚：氧氧化还原酶），还表现出其他相关活性。最近，有报道称市售蘑菇酪氨酸酶制剂催化了一种新反应，即2,6 - 二甲氧基烯丙基苯酚氧化转化为其亚甲基醌（E. S. 克罗尔和J. L. 博尔顿，1997年，《化学生物学相互作用》104卷，11 - 27页）。由于该反应涉及不寻常的1,6 - 氧化而非传统的1,4 - 氧化，我们更仔细地重新研究了此反应。蘑菇酪氨酸酶制剂的邻二酚氧化酶活性和二甲氧基烯丙基苯酚氧化酶活性在Sephacryl S - 200柱上的尺寸排阻色谱中表现出不同的迁移率。在旋转聚焦细胞中进行制备性等电聚焦时也观察到了类似行为。不同的蘑菇酪氨酸酶制剂这两种活性的比例各不相同，进一步证实它们是由两种不同的酶引起的。天然聚丙烯酰胺凝胶电泳后对凝胶进行活性染色显示这两种活性具有不同的迁移率。表现出二甲氧基烯丙基苯酚氧化酶活性的蛋白带也可以用丁香醛连氮染色，丁香醛连氮是漆酶（EC 1.10.3.2，对二酚：氧氧化还原酶）的一种著名底物。两种昆虫酚氧化酶以其广泛的底物特异性而闻名，但未能将二甲氧基烯丙基苯酚氧化到可检测的程度，从而证实典型的邻二酚氧化酶缺乏氧化二甲氧基烯丙基苯酚的能力。另一方面，已知能将丁香醛连氮转化为其亚甲基醌衍生物的漆酶能轻易地从二甲氧基烯丙基苯酚产生亚甲基醌。因此得出结论，作为市售蘑菇酪氨酸酶制剂中的污染物存在的漆酶——而非酪氨酸酶（邻二酚氧化酶）——是催化二甲氧基烯丙基苯酚向其相应亚甲基醌新转化的酶。
