Probing Ligand Exchange in the P450 Enzyme CYP121 from Mycobacterium tuberculosis: Dynamic Equilibrium of the Distal Heme Ligand as a Function of pH and Temperature.
Probing Ligand Exchange in the P450 Enzyme CYP121 from Mycobacterium tuberculosis: Dynamic Equilibrium of the Distal Heme Ligand as a Function of pH and Temperature.
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DOI：
10.1021/jacs.7b08911
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发表时间：
2017-12-06
影响因子：
15
通讯作者：
Liu A
中科院分区：
文献类型：
--
作者：
Fielding AJ;Dornevil K;Ma L;Davis I;Liu A
CYP121是一种来自结核分枝杆菌的细胞色素P450酶，它催化其环二酪氨酸底物（cYY）的芳香基团之间形成C-C键。CYP121与cYY配合物的晶体结构表明，在酶-底物配合物中，溶剂衍生的配体仍然与铁离子结合。而在普遍接受的P450机制中，活性位点的初级底物结合触发溶剂衍生配体的释放，引发金属中心还原和随后的O2结合。在这里，我们使用氰化钠来探测酶和酶-底物复合物的金属配体交换。用紫外可见光谱、EPR光谱、ENDOR光谱和x射线晶体学对氰基加合物进行了表征。观察到与无配体酶相比，氰化物与酶-底物复合物结合的亲和力增加了100倍。与二元[CYP121(cYY)]配合物的结构相比，[CYP121(cYY)CN]三元配合物的晶体结构在活性位点表现出底物的重排。瞬态动力学研究表明，cYY结合产生较低的二阶速率常数（kon (CN)），但更稳定的氰化物加合物，其koff （CN）速率降低了3个数量级。酶和酶-底物复合物的多个高自旋和低自旋物质之间存在动态平衡，该平衡对pH和温度的变化都很敏感。我们的数据揭示了酶的溶剂衍生配体的化学和物理性质，这将有助于了解催化机制的初始步骤。
CYP121 is a cytochrome P450 enzyme from Mycobacterium tuberculosis that catalyzes the formation of a C–C bond between the aromatic groups of its cyclodityrosine substrate (cYY). The crystal structure of CYP121 in complex with cYY reveals that the solvent-derived ligand remains bound to the ferric ion in the enzyme–substrate complex. Whereas in the generally accepted P450 mechanism, binding of the primary substrate in the active-site triggers the release of the solvent-derived ligand, priming the metal center for reduction and subsequent O2 binding. Here we employed sodium cyanide to probe the metal–ligand exchange of the enzyme and the enzyme–substrate complex. The cyano adducts were characterized by UV–vis, EPR, and ENDOR spectroscopies and X-ray crystallography. A 100-fold increase in the affinity of cyanide binding to the enzyme–substrate complex over the ligand-free enzyme was observed. The crystal structure of the [CYP121(cYY)CN] ternary complex showed a rearrangement of the substrate in the active-site, when compared to the structure of the binary [CYP121(cYY)] complex. Transient kinetic studies showed that cYY binding resulted in a lower second-order rate constant (kon (CN)) but a much more stable cyanide adduct with 3 orders of magnitude slower koff (CN) rate. A dynamic equilibrium between multiple high- and low-spin species for both the enzyme and enzyme–substrate complex was also observed, which is sensitive to changes in both pH and temperature. Our data reveal the chemical and physical properties of the solvent-derived ligand of the enzyme, which will help to understand the initial steps of the catalytic mechanism.
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影响因子：
2.9
作者：
GOOD, NE;WINGET, GD;SINGH, RMM
通讯作者：
SINGH, RMM
无底物细胞色素P450中轴向水结扎的强度取决于同工型。
影响因子：
2.9
作者：
Conner, Kip P.;Schimpf, Alina M.;Cruce, Alex A.;McLean, Kirsty J.;Munro, Andrew W.;Frank, Daniel J.;Krzyaniak, Matthew D.;de Montellano, Paul Ortiz;Bowman, Michael K.;Atkins, William M.
通讯作者：
Atkins, William M.
影响因子：
24.3
作者：
Kliiman, K.;Altraja, A.
通讯作者：
Altraja, A.
影响因子：
7.3
作者：
Kavanagh ME;Coyne AG;McLean KJ;James GG;Levy CW;Marino LB;de Carvalho LP;Chan DS;Hudson SA;Surade S;Leys D;Munro AW;Abell C
通讯作者：
Abell C
DOI：
10.1107/s0907444909052925
发表时间：
2010-02
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Adams PD;Afonine PV;Bunkóczi G;Chen VB;Davis IW;Echols N;Headd JJ;Hung LW;Kapral GJ;Grosse-Kunstleve RW;McCoy AJ;Moriarty NW;Oeffner R;Read RJ;Richardson DC;Richardson JS;Terwilliger TC;Zwart PH
通讯作者：
Zwart PH