Steady-state and transient kinetic analyses of taurine/α-ketoglutarate dioxygenase:: Effects of oxygen concentration, alternative sulfonates, and active-site variants on the feIV-oxo intermediate
Steady-state and transient kinetic analyses of taurine/α-ketoglutarate dioxygenase:: Effects of oxygen concentration, alternative sulfonates, and active-site variants on the feIV-oxo intermediate
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DOI：
10.1021/bi048746n
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发表时间：
2005-03-15
期刊：
影响因子：
2.9
通讯作者：
Hausinger, RP
中科院分区：
文献类型：
--
作者：
Grzyska, PK;Ryle, MJ;Hausinger, RP
牛磺酸/α-酮戊二酸（α XG）双加氧酶（TauD）是一种原型α KG依赖性羟化酶，是一种非血红素单核Fe-II酶，可将α KG的氧化脱羧作用与牛磺酸转化为氨基乙醛和亚硫酸盐偶联。牛磺酸-α KG-Fe（II）TauD的晶体结构是已知的，并且光谱研究已经从动力学上定义了催化的早期步骤，并确定了高自旋Fe-IV-氧代反应中间体。目前的分析扩展了我们的理解TauD催化通过调查的稳态和瞬态动力学的野生型和变异形式的酶与牛磺酸和替代磺酸盐。在活性位点周围的残基处取代的TauD蛋白质显示基于其形成与厌氧Fe-II-α KG螯合复合物相关的诊断发色团的能力适当折叠，并且在随后与氧反应时产生酪氨酰自由基。突变蛋白的稳态研究证实了His 70和Arg 270在结合牛磺酸磺酸部分中的重要性，并表明Asn 95参与识别底物胺基团。N97 A和S158 A变体可能经历疏水性的增加和底物结合口袋的扩张，因此与野生型TauD相比，它们对戊磺酸的Km降低。氧与牛磺酸-α KG-Fe（II）TauD反应的停流紫外-可见光谱检查证实了归因于Fe-IV-氧代形成（k（1））、底物羟基化后漂白为Fe-II状态（k（2））、过量底物再结合（k（3））的最小三步反应序列，并表明没有一个步骤显示出可检测的溶剂k（H）/k（D）同位素效应。这表明，没有质子参与铁IV-氧代形成的速率决定步骤，与血红素铁加氧酶相反。Fe-IV-氧代物质可能用于转化替代底物戊磺酸和3-N-吗啉代丙磺酸;然而，由于k（1）/k（2）比值降低，未检测到该光谱中间体。对于牛磺酸，k（1）取决于氧浓度，允许计算该不可逆反应的二级速率常数为1.58 × 10（5）M-1 s（-1）。TauD变体的停流分析为蛋白质环境如何影响Fe-IV-oxo形成和衰变的速率提供了一些见解。在N95 D或N95 A变体中未检测到Fe-IV-氧代物质，因为k（1）/k（2）比值降低，可能与六配位至五配位金属位点的底物依赖性转化降低有关。
Taurine/alpha-ketoglutarate (alpha XG) dioxygenase (TauD), an archetype alpha KG-dependent hydroxylase, is a non-heme mononuclear Fe-II enzyme that couples the oxidative decarboxylation of alpha KG with the conversion of taurine to aminoacetaldehyde and sulfite. The crystal structure of taurine-alpha KG-Fe(II)TauD is known, and spectroscopic studies have kinetically defined the early steps in catalysis and identified a high-spin Fe-IV-oxo reaction intermediate. The present analysis extends our understanding of TauD catalysis by investigating the steady-state and transient kinetics of wild-type and variant forms of the enzyme with taurine and alternative sulfonates. TauD proteins substituted at residues surrounding the active site were shown to fold properly based on their abilities to form a diagnostic chromophore associated with the anaerobic Fe-II-alpha KG chelate complex and to generate a tyrosyl radical upon subsequent reaction with oxygen. Steady-state studies of mutant proteins confirmed the importance of His 70 and Arg 270 in binding, the sulfonate moiety of taurine and indicated the participation of Asn 95 in recognizing the substrate amine group. The N97A and S158A variants are likely to undergo an increase in hydrophobicity and expansion of the substrate-binding pocket, thus accounting for their decreased K-m toward pentanesulfonic acid compared to wild-type TauD. Stopped-flow UV-visible spectroscopic examination of the reaction of oxygen with taurine-alpha KG-Fe(II)TauD confirmed a minimal three-step sequence of reactions attributed to Fe-IV-oxo formation (k(1)), bleaching to the Fe-II state upon substrate hydroxylation (k(2)), rebinding of excess substrates (k(3)) and indicated that none of the steps exhibit detectable solvent k(H)/k(D) isotope effects. This demonstrates that no protons are involved in the rate-determining step of Fe-IV-oxo formation, in contrast to heme iron oxygenases. The Fe-IV-oxo species is likely to be utilized in conversion of the alternative substrates pentanesulfonic acid and 3-N-morpholinopropanesulfonic acid; however, this spectroscopic intermediate was not detected because of the decreased k(1)/k(2) ratio. With taurine, k(1), was shown to depend on the oxygen concentration allowing calculation of a second-order rate constant of 1.58 x 10(5) M-1 s(-1) for this irreversible reaction. Stopped-flow analyses of TauD variants provided several insights into how the protein environment influences the rates of Fe-IV-oxo formation and decay. The Fe-IV-oxo species was not detected in the N95D or N95A variants because of a reduced k(1)/k(2) ratio, likely related to a decreased substrate-dependent conversion of the six-coordinate to five-coordinate metal site.