Single-turnover kinetics of homoprotocatechuate 2,3-dioxygenase
Single-turnover kinetics of homoprotocatechuate 2,3-dioxygenase
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DOI：
10.1021/bi048690x
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发表时间：
2004-12-07
期刊：
影响因子：
2.9
通讯作者：
Lipscomb, JD
中科院分区：
文献类型：
--
作者：
Groce, SL;Miller-Rodeberg, MA;Lipscomb, JD
从褐色短杆菌中分离的高原儿茶酸2，3-双加氧酶利用活性位点Fe（II）和O-2催化底物芳环的近端extradiol裂解。与开环双加氧酶家族的其他成员相比，extradiol双加氧酶催化循环的瞬时动力学一直难以研究，因为铁几乎是无色的并且EPR沉默。在这里，它表明，反应周期动力学可以通过利用替代底物4-硝基儿茶酚（4 NC），这也是裂解在近端extradiol位置进行监测。由于电离、环境变化和环裂解而发生的4 NC的光谱变化允许研究反应的底物结合和产物形成阶段。结果表明，底物结合发生在一个四步的过程中可能涉及绑定到两个电离状态的酶在不同的速率。在活性位点中的单阴离子4 NC的初始快速结合之后，发生与Fe（II）的较慢结合和向双阴离子形式的转化。结合的双阴离子4 NC反应迅速与O-2在四个额外的步骤，显然发生顺序。根据中间体的光学性质，这些步骤被假设为O-2结合到铁，异构化所产生的复合物，开环，和产品的释放。天然底物似乎形成相同的中间体，但具有大得多的速率常数。这些是第一个短暂的中间体被报道的extradiol双加氧酶反应。
Homoprotocatechuate 2,3-dioxygenase isolated from Brevibacterium fuscum utilizes an active site Fe(II) and O-2 to catalyze proximal extradiol cleavage of the substrate aromatic ring. In contrast to other members of the ring cleaving dioxygenase family, the transient kinetics of the extradiol dioxygenase catalytic cycle have been difficult to study because the iron is nearly colorless and EPR silent. Here, it is shown that the reaction cycle kinetics can be monitored by utilizing the alternative substrate 4-nitrocatechol (4NC), which is also cleaved in the proximal extradiol position. Changes in the optical spectrum of 4NC occurring as a result of ionization, environmental changes, and ring cleavage allow both the substrate binding and product formation phases of the reaction to be studied. It is shown that substrate binding occurs in a four-step process probably involving binding to two ionization states of the enzyme at different rates. Following an initial rapid binding of the monoanionic 4NC in the active site, slower binding to the Fe(II) and conversion to the dianionic form occur. The bound dianionic 4NC reacts rapidly with O-2 in four additional steps, apparently occurring in sequence. On the basis of the optical properties of the intermediates, these steps are hypothesized to be O-2 binding to the iron, isomerization of the resulting complex, ring opening, and product release. The natural substrate appears to form the same intermediates but with much larger rate constants. These are the first transient intermediates to be reported for an extradiol dioxygenase reaction.