Rates of the phthalate dioxygenase reaction with oxygen are dramatically increased by interactions with phthalate and phthalate oxygenase reductase.
Rates of the phthalate dioxygenase reaction with oxygen are dramatically increased by interactions with phthalate and phthalate oxygenase reductase.
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邻苯二甲酸酯双加氧酶与氧的反应速率通过与邻苯二甲酸酯和邻苯二甲酸酯加氧酶还原酶的相互作用而显着增加。
DOI：
10.1021/bi0490587
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Ballou,DavidP
中科院分区：
文献类型：
--
作者：
Tarasev,Michael;Rhames,Frank;Ballou,DavidP
邻苯二甲酸酯双加氧酶系统催化邻苯二甲酸酯的二羟基化形成邻苯二甲酸二氢二醇（DHD），其具有两个组分：邻苯二甲酸酯双加氧酶（PDO），其是每个单体具有一个Rieske型[2Fe-2S]和一个Fe（II）中心的多聚体;以及邻苯二甲酸酯双加氧酶还原酶（PDR），其含有黄素单甘肽（FMN）和植物样铁氧还蛋白[2Fe-2S]中心。PDR负责将电子从NADH转移到PDO的Rieske中心，Rieske中心为底物的氧化提供电子给单核中心。在单核金属位点（PDO-APO）缺少Fe（II）的还原PDO（PDOred）与O2反应缓慢（125 μM O2和22 °C时为1.4 × 10-3s-1），可能与Rieske中心直接反应。邻苯二甲酸酯和/或PDRox与还原的PDO-APO的结合增加了Rieske中心与O2的反应性。当不存在PDR或邻苯二甲酸酯时，天然PDOred[在单核位点含有Fe（II）]中Rieske中心的氧化分两个阶段发生（125 mM O2，23 °C下为0.1 s-1和0.1 s-1），两者都比不存在Fe（II）时快得多，可能是因为在这种情况下O2与单核Fe（II）反应。加入PDRox到原生PDOred中导致大部分Rieske中心在5 s-1被氧化，加入邻苯二甲酸酯导致约70%的反应在42 s-1进行。当PDRox和邻苯二甲酸酯同时存在时，大部分PDOred（约80−85%）在42 s-1时氧化，其余的在105 s-1时氧化。因此，邻苯二甲酸酯或PDRox与PDO redeach的结合导致PDO与O2的更大反应性。底物和PDR两者的存在是协同的，使得PDO完全具有催化活性。一个模型，解释所观察到的效果，并讨论了PDO亚基协同性。有人建议，在氧化过程中减少PDO，每个两个Rieske中心在单独的亚基转移一个电子的Fe（II）单核中心的第三个亚基。这种解释是一致的，观察到的多相动力学的Rieske中心的氧化，并正在进一步测试的产品分析实验。
The phthalate dioxygenase system, which catalyzes the dihydroxylation of phthalate to form itscis-dihydrodiol (DHD), has two components: phthalate dioxygenase (PDO), a multimer with one Rieske-type [2Fe-2S] and one Fe(II) center per monomer, and phthalate dioxygenase reductase (PDR), which contains flavin mononucleotide (FMN) and a plant-like ferredoxin [2Fe-2S] center. PDR is responsible for transferring electrons from NADH to the Rieske center of PDO, and the Rieske center supplies electrons to the mononuclear center for the oxygenation of substrate. Reduced PDO (PDOred) that lacks Fe(II) at the mononuclear metal site (PDO-APO) reacts slowly with O2(1.4 × 10-3s-1at 125 μM O2and 22 °C), presumably in a direct reaction with the Rieske center. Binding of phthalate and/or PDRoxto reduced PDO-APO increases the reactivity of the Rieske center with O2. When no PDR or phthalate is present, the oxidation of the Rieske center in native PDOred[which contains Fe(II) at the mononuclear site] occurs in two phases (∼1 and 0.1 s-1at 125 mM O2, 23 °C), both much faster than in the absence of Fe(II), presumably because in this case O2reacts at the mononuclear Fe(II). Addition of PDRoxto native PDOredresulted in a large fraction of the Rieske center being oxidized at 5 s-1, and the addition of phthalate resulted in about 70% of the reaction proceeding at 42 s-1. With both PDRoxand phthalate present, most of the PDOred(approximately 80−85%) oxidizes at 42 s-1, with the remaining oxidizing at ∼5 s-1. Thus, the binding of phthalate or PDRoxto PDOredeach results in greater reactivity of PDO with O2. The presence of both the substrate and PDR was synergistic, making PDO fully catalytically active. A model that explains the observed effects is presented and discussed in terms of PDO subunit cooperativity. It is proposed that, during oxidation of reduced PDO, each of two Rieske centers on separate subunits transfers an electron to the Fe(II) mononuclear center on a third subunit. This explanation is consistent with the observed multiphasic kinetics of the oxidation of the Rieske center and is being further tested by product analysis experiments.