Structural Basis for Substrate and Oxygen Activation in Homoprotocatechuate 2,3-Dioxygenase: Roles of Conserved Active Site Histidine 200.
Structural Basis for Substrate and Oxygen Activation in Homoprotocatechuate 2,3-Dioxygenase: Roles of Conserved Active Site Histidine 200.
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DOI：
10.1021/acs.biochem.5b00709
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发表时间：
2015-09-01
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：
Kovaleva EG;Rogers MS;Lipscomb JD
动力学和光谱研究表明，保守的活性位点残基His 200的extradiol环裂解homoprotocatechuate 2，3-双加氧酶（FeHPCD）从短杆菌fuscum是关键的有效催化。这个残基所发挥的作用在这里探测通过分析的稳态动力学，pH依赖性，和X-射线晶体结构的FeHPCD位置200变体His 200 Asn，His 200 Gln，和His 200 Glu单独和复杂的三个儿茶酚底物（homoprotocatechuate，4-sulfonylcatechol，和4-nitrocatechol）具有不同的诱导能力的取代基。在1.35 - 1.75 nm分辨率下解析的结构表明，与野生型酶及其类似复合物的结构相比，这些变体的总体活性位点结构或底物结合模式基本上没有变化。这表明，最大的50倍降低kcat的环裂解，pH依赖性的急剧变化，和4-硝基儿茶酚的环裂解的开关环氧化的FeHPCD变体可以具体归因于改变的第二球残基和基板的性能。结果表明，质子转移是催化所必需的，并且当底物提供质子并且His 200用作催化剂时，它最有效地发生。然而，在没有可用的底物质子的情况下，可以利用蛋白质中的确定的质子转移途径。在空间体积和电荷的位置200的残基的变化出现能够改变催化的限速步骤，也许，反应物种的性质。
Kinetic and spectroscopic studies have shown that the conserved active site residue His200 of the extradiol ring-cleaving homoprotocatechuate 2,3-dioxygenase (FeHPCD) from Brevibacterium fuscum is critical for efficient catalysis. The roles played by this residue are probed here by analysis of the steady state kinetics, pH dependence, and X-ray crystal structures of the FeHPCD position 200 variants His200Asn, His200Gln, and His200Glu alone and in complex with three catecholic substrates (homoprotocatechuate, 4-sulfonylcatechol, and 4-nitrocatechol) possessing substituents with different inductive capacity. Structures solved at 1.35 –1.75 Å resolution show that there is essentially no change in overall active site architecture or substrate binding mode for these variants when compared to the structures of the wild type enzyme and its analogous complexes. This shows that the maximal 50-fold decrease in kcat for ring cleavage, the dramatic changes in pH dependence, and the switch from ring cleavage to ring oxidation of 4-nitrocatechol by the FeHPCD variants can be attributed specifically to the properties of the altered second sphere residue and the substrate. The results suggest that proton transfer is necessary for catalysis, and that it occurs most efficiently when the substrate provides the proton and His200 serves as a catalyst. However, in the absence of an available substrate proton, a defined proton-transfer pathway in the protein can be utilized. Changes in steric bulk and charge of the residue at position 200 appear capable of altering the rate-limiting step in catalysis, and perhaps, the nature of the reactive species.