Kinetic evaluation of catalase and peroxygenase activities of tyrosinase.
Kinetic evaluation of catalase and peroxygenase activities of tyrosinase.
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DOI：
10.1021/bi048908f
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发表时间：
2004-08
期刊：
影响因子：
2.9
通讯作者：
S. Yamazaki;Chiyuki Morioka;S. Itoh
中科院分区：
文献类型：
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作者：
S. Yamazaki;Chiyuki Morioka;S. Itoh
酪氨酸酶是一种含有偶联双核铜活性位点（3型铜）的铜单加氧酶，它以O(2)为氧化剂催化酚类的氧化（酚酶活性）和儿茶酚类的脱氢（儿茶酚酶活性）。在本研究中，利用电流型O(2)和H(2)O(2)传感器对蘑菇酪氨酸酶的过氧化氢酶活性(H(2)O(2)转化为（1/2）O(2)和H（2)O(2) O）和过氧酶活性(H（2)O(2)依赖底物的氧化）进行了动力学研究。在初始厌氧条件下，在0.1 M磷酸盐缓冲液（pH 7.0）中，在初始厌氧条件下，在酪氨酸酶的催化量下，通过监测H(2)O(2)产生O(2)的初始速率来检测过氧化氢酶的活性。研究发现，蘑菇酪氨酸酶过氧化氢酶活性比软体动物血青素高3个数量级。酪氨酸酶过氧化氢酶活性较高可能是由于H(2)O(2)更容易接近酪氨酸酶的双核铜位点。蘑菇酪氨酸酶也首次被证实能催化苯酚与H(2)O(2)（过氧酶活性）的氧化反应。通过监测好氧条件下0.5 M硼酸缓冲液（pH 7.0）中H(2)O(2)的消耗速率，对该反应进行了动力学研究。一系列对取代的苯酚在与H(2)O(2)的过氧酶反应中的取代效应与与O(2)的苯酚酶反应中的取代效应相似，以及与过氘化底物(p-Cl-C(6)D(4)OH vs p-Cl-C（6)H(4)OH）没有动力学氘同位素效应，清楚地表明两种体系中苯酚的氧化机制是相同的，即：氧酪氨酸酶的(微-eta（2):eta(2)-过氧）二铜（II）中间体的亲电芳香取代反应。
Tyrosinase is a copper monooxygenase containing a coupled dinuclear copper active site (type-3 copper), which catalyzes oxygenation of phenols (phenolase activity) as well as dehydrogenation of catechols (catecholase activity) using O(2) as the oxidant. In this study, catalase activity (conversion of H(2)O(2) to (1/2)O(2) and H(2)O) and peroxygenase activity (H(2)O(2)-dependent oxygenation of substrates) of mushroom tyrosinase have been examined kinetically by using amperometric O(2) and H(2)O(2) sensors. The catalase activity has been examined by monitoring the initial rate of O(2) production from H(2)O(2) in the presence of a catalytic amount of tyrosinase in 0.1 M phosphate buffer (pH 7.0) at 25 degrees C under initially anaerobic conditions. It has been found that the catalase activity of mushroom tyrosinase is three-order of magnitude greater than that of mollusk hemocyanin. The higher catalase activity of tyrosinase could be attributed to easier accessibility of H(2)O(2) to the dinuclear copper site of tyrosinase. Mushroom tyrosinase has also been demonstrated for the first time to catalyze oxygenation reaction of phenols with H(2)O(2) (peroxygenase activity). The reaction has been investigated kinetically by monitoring the H(2)O(2) consumption rate in 0.5 M borate buffer (pH 7.0) under aerobic conditions. Similarity of the substituent effects of a series of p-substituted phenols in the peroxygenase reaction with H(2)O(2) to those in the phenolase reaction with O(2) as well as the absence of kinetic deuterium isotope effect with a perdeuterated substrate (p-Cl-C(6)D(4)OH vs p-Cl-C(6)H(4)OH) clearly demonstrated that the oxygenation mechanisms of phenols in both systems are the same, that is, the electrophilic aromatic substitution reaction by a (micro-eta(2):eta(2)-peroxo)dicopper(II) intermediate of oxy-tyrosinase.