Naish-Byfield S, Riley P A
Department of Biology and Biochemistry, Brunel University, Uxbridge, Middx, U.K.
Biochem J. 1992 Nov 15;288 ( Pt 1)(Pt 1):63-7. doi: 10.1042/bj2880063.
The purity of commercially available mushroom tyrosinase was investigated by non-denaturing PAGE. Most of the protein in the preparation migrated as a single band under these conditions. This band contained both tyrosinase and dopa oxidase activity. No other activity of either classification was found in the preparation. Oxygen consumption by tyrosinase during oxidation of the monohydric phenol substrates tyrosine and 4-hydroxyanisole (4HA) was monitored by oximetry in order to determine the stoichiometry of the reactions. For complete oxidation, the molar ratio of oxygen: 4HA was 1:1. Under identical conditions, oxidation of tyrosine required 1.5 mol of oxygen/mol of tyrosine. The additional oxygen uptake during tyrosine oxidation is due to the internal cyclization of dopaquinone to form cyclodopa, which undergoes a redox reaction with dopaquinone to form dopachrome and dopa, which is then oxidized by the enzyme, leading to an additional 0.5 mol of oxygen/mol of original substrate. Oxygen consumption for complete oxidation of 200 nmol of 4HA was constant over a range of concentrations of tyrosinase of 33-330 units/ml of substrate. The maximum rate of reaction was directly proportional to the concentration of tyrosinase, whereas the length of the lag phase decreased non-linearly with increasing tyrosinase concentration. Activation of the enzyme by exposure to citrate was not seen, nor was the lag phase abolished by exposure of the enzyme to low pH. Michaelis-Menten analysis of tyrosinase in which the lag phase is abolished by pre-exposure of the enzyme to a low concentration of dithiothreitol gave Km values for tyrosine and 4HA of 153 and 20 microM respectively.
采用非变性聚丙烯酰胺凝胶电泳（PAGE）对市售蘑菇酪氨酸酶的纯度进行了研究。在此条件下，制剂中的大多数蛋白质迁移为单一谱带。该谱带同时含有酪氨酸酶和多巴氧化酶活性。在制剂中未发现其他任何一种类型的活性。通过血氧测定法监测酪氨酸酶在一元酚底物酪氨酸和4 - 羟基苯甲醚（4HA）氧化过程中的耗氧量，以确定反应的化学计量关系。对于完全氧化，氧气与4HA的摩尔比为1:1。在相同条件下，酪氨酸氧化需要每摩尔酪氨酸消耗1.5摩尔氧气。酪氨酸氧化过程中额外的氧气摄取是由于多巴醌的内环化形成环多巴，环多巴与多巴醌发生氧化还原反应形成多巴色素和多巴，然后多巴被该酶氧化，导致每摩尔原始底物额外消耗0.5摩尔氧气。在底物浓度为33 - 330单位/毫升的酪氨酸酶浓度范围内，200纳摩尔4HA完全氧化的耗氧量是恒定的。最大反应速率与酪氨酸酶浓度成正比，而延迟期的长度随酪氨酸酶浓度增加呈非线性下降。未观察到通过柠檬酸盐处理激活该酶的情况，将酶暴露于低pH值也未消除延迟期。对酪氨酸酶进行米氏分析时，通过将酶预先暴露于低浓度的二硫苏糖醇来消除延迟期，结果表明酪氨酸和4HA的米氏常数（Km）分别为153和20微摩尔。
