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首页> 外文期刊>Biochemistry >Kinetic and CD/MCD Spectroscopic Studies of the Atypical, Three-His-Ligated, Non-Heme Fe2+ Center in Diketone Dioxygenase: The Role of Hydrophilic Outer Shell Residues in Catalysis
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Kinetic and CD/MCD Spectroscopic Studies of the Atypical, Three-His-Ligated, Non-Heme Fe2+ Center in Diketone Dioxygenase: The Role of Hydrophilic Outer Shell Residues in Catalysis

机译：二酮双加氧酶中非典型的，三组氨酸连接的，非血红素Fe2 +中心的动力学和CD / MCD光谱研究：亲水性外壳残留物在催化中的作用

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摘要

Diketone cleaving enzyme (Dke1) is a dioxygenase with an atypical, three-histidine-ligated, mono-nnuclear non-heme Fen2þ center. To assess the role in enzyme catalysis of the hydrophilic residues in the active sitenpocket, residues Glu98, Arg80, Tyr70, and Thr107 were subjected to mutational analysis. Steady state and pre-nsteady state kinetics indicated a role for Glu98 in promoting both substrate binding and O2 reduction.nAdditionally, the Glu98 substitution eliminated the pH dependence of substrate binding (kcatnappn/KMappn-pHnprofile) present in wild-typeDke1 (pKa=6.3(0.4 and 8.4(0.4).MCD spectroscopy revealed that theGlu98nGln mutation leads to the conversion of the six-coordinate (6C) resting Fen2þ center present in the wild-typenenzyme at pH7.0 to amixture of five-coordinate (5C) and 6C sites. The 6C geometrywas restored with a pHshiftnto 9.5 which also resulted in ligand field (LF) energy splittings identical to that found for wild-type (WT) Dke1 atnpH 9.5. In WT Dke1, these LF transitions are shifted up in energy by ∼300 cm-1nat pH 9.5 relative to pH 7.0.nThese data, combined with CD pH titrations which reveal a pKa of ∼8.2forrestingWTDke1andtheGlu98nGln variant, indicate the deprotonation of a metal-ligated water. Together, the kinetic and spectroscopic datanreveal a stabilizing effect ofGlu98 on the 6C geometry of themetal center, priming it for substrate ligation.Arg80nand Tyr70 are shown to promote O2 reduction, while Thr107 stabilizes the Fe(II) cofactor.
机译：二酮裂解酶（Dke1）是一种双加氧酶，具有非典型的三组氨酸连接的单核非血红素Fen2þ中心。为了评估活性位点袋中亲水残基在酶催化中的作用，对残基Glu98，Arg80，Tyr70和Thr107进行了突变分析。稳态和稳态前动力学表明Glu98在促进底物结合和O2还原中都起作用.n此外，Glu98取代消除了野生型Dke1（pKa = 6.3）中底物结合的pH依赖性（kcatnappn / KMappn-pHnprofile）。 （0.4和8.4（0.4）.MCD光谱显示，Glu98nGln突变导致存在于pH7.0的野生型酶中的六坐标（6C）静止Fen2þ中心转变为五坐标（5C）和6C的混合物用pHshiftnto 9.5恢复6C的几何形状，这也导致配体场（LF）的能量分裂与野生型（WT）Dke1在pH为9.5时发现的相同。在WT Dke1中，这些LF跃迁的能量向上移动了〜。 300 cm-1nat pH 9.5相对于pH 7.0.n这些数据，结合CD pH滴定显示，对于残留的WTDke1和Glu98nGln变体，pKa约为8.2，表明与金属连接的水具有去质子性，动力学和光谱数据共同显示了一个稳定剂Glu98对金属中心6C几何构型的活化作用，引发了底物的连接。Arg80n和Tyr70可以促进O2还原，而Thr107可以稳定Fe（II）辅因子。

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