Benzene-induced uncoupling of naphthalene dioxygenase activity and enzyme inactivation by production of hydrogen peroxide
Benzene-induced uncoupling of naphthalene dioxygenase activity and enzyme inactivation by production of hydrogen peroxide
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DOI：
10.1128/jb.181.9.2719-2725.1999
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发表时间：
1999-05-01
影响因子：
3.2
通讯作者：
Lee, K
中科院分区：
文献类型：
--
作者：
Lee, K
萘双加氧酶（Naphthalene dioxygenase，NDO）是一种多组分酶系统，它通过消耗NAD（P）H中的O-2和两个电子，将萘氧化为（+）-cis-（1 R，2S）-1，2-dihydroxy-1，2-dihydroxy-naphthalene。在苯的存在下，NADH氧化和O-2利用部分解耦从底物氧化。大约40 - 50%的消耗的O-2被检测为过氧化氢。苯依赖的O-2消耗速率随时间的推移而降低，但在NDO消耗O-2的过程中加入过氧化氢酶可部分提高苯依赖的O-2消耗速率。详细的实验表明，在过氧化氢清除剂的存在下，O-2的消耗总量和苯诱导的O-2消耗速率增加，并进一步添加NDO的末端加氧酶组分（ISPNAP）。动力学研究表明，ISPNAP是不可逆的失活反应中，含有苯，但失活被解除到一个高度的存在下，过氧化氢酶和0.1 mM亚铁离子的存在下，部分缓解。苯和萘反应的ISPNAP给出了几乎相同的可见吸收光谱。此外，以0.1至0.6 mM的范围添加到反应混合物中的过氧化氢使含有单核铁的还原ISPNAP失活。这些结果表明，在解偶联反应过程中释放的过氧化氢既作为苯依赖的O-2消耗的抑制剂，又作为ISPNAP的灭活剂。有人提出，ISPNAP的不可逆失活是通过形成强氧化剂羟基自由基（（OH）-O-）的Fenton型反应发生的，从过氧化氢的反应，vith亚铁单核铁在活性位点。此外，当[C-14]苯作为底物时，检测到由NDO形成的顺式苯1，2-二氢二醇。该结果表明，NDO还将痕量的苯与O-2消耗和NADH氧化偶联。
Naphthalene dioxygenase (NDO) is a multicomponent enzyme system that oxidizes naphthalene to (+)-cis-(1R,2S)-1,2-dihydroxy-1,2-dihydronaphthalene with consumption of O-2 and two electrons from NAD(P)H. In the presence of benzene, NADH oxidation and O-2 utilization were partially uncoupled from substrate oxidation. Approximately 40 to 50% of the consumed O-2 was detected as hydrogen peroxide. The rate of benzene-dependent O-2 consumption decreased with time, but it was partially increased by the addition of catalase in the course of the O-2 consumption by NDO. Detailed experiments showed that the total amount of O-2 consumed and the rate of benzene-induced O-2 consumption increased in the presence of hydrogen peroxide-scavenging agents, and further addition of the terminal oxygenase component (ISPNAP) of NDO. Kinetic studies showed that ISPNAP was irreversibly inactivated in the reaction that contained benzene, but the inactivation was relieved to a high degree in the presence of catalase and partially relieved in the presence of 0.1 mM ferrous ion. Benzene- and naphthalene-reacted ISPNAP gave almost identical visible absorption spectra. In addition, hydrogen peroxide added at a range of 0.1 to 0.6 mM to the reaction mixtures inactivated the reduced ISPNAP containing mononuclear iron. These results show that hydrogen peroxide released during the uncoupling reaction acts both as an inhibitor of benzene-dependent O-2 consumption and as an inactivator of ISPNAP. It is proposed that the irreversible inactivation of ISPNAP occurs by a Fenton-type reaction which forms a strong oxidizing agent, hydroxyl radicals ((OH)-O-.), from the reaction of hydrogen peroxide,vith ferrous mononuclear iron at the active site. Furthermore, when [C-14]benzene was used as the substrate, cis-benzene 1,2-dihydrodiol formed by NDO was detected. This result shows that NDO also couples a trace amount of benzene to both O-2 consumption and NADH oxidation.