Spin equilibrium and O₂-binding kinetics of Mycobacterium tuberculosis CYP51 with mutations in the histidine-threonine dyad.
Spin equilibrium and O₂-binding kinetics of Mycobacterium tuberculosis CYP51 with mutations in the histidine-threonine dyad.
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DOI：
10.1016/j.jinorgbio.2014.03.017
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发表时间：
2014-07
影响因子：
3.9
通讯作者：
Hackett, John C.
中科院分区：
文献类型：
--
作者：
Jennings, Gareth K.;Modi, Anuja;Elenewski, Justin E.;Ritchie, Caroline M.;Thuy Nguyen;Ellis, Keith C.;Hackett, John C.
CYP 51酶中“酸-醇对”的酸性残基被组氨酸均匀取代。在此，我们采用结核分枝杆菌（mt）酶作为模型系统，以研究这些残基的作用，微调血红素构象，铁自旋状态，形成和衰变的氧亚铁酶。使用UV-Vis和共振拉曼光谱来询问mtCYP 51和T260 A、T260 V和H259 A突变体的性质。证据支持这些突变引起血红素环境的全面变化。血红素铁自旋状态对底物二氢羊毛甾醇（DHL）的结合具有不同的敏感性。DHL和克霉唑扰乱血红素乙烯基和丙酸酯取代基的局部环境。DHL-酶复合物的分子动力学（MD）模拟支持所观察到的扰动是由于DHL结合模式的变化。此外，使用停流方法测量氧化亚铁形成的速率。这些研究表明，HT突变和DHL都能调节含氧亚铁形成的速率。巧合的是，与H259 A突变体-DHL复合物的结合率约为mtCYP 51的4倍，这一现象被预测为由于突变体中H259-E173离子对的丢失而产生额外的扩散通道。含氧亚铁酶自动氧化速率相对恒定，除了T260 V-DHL复合物。MD模拟导致我们推测，这种行为可能是由于扭曲的血红素大环的基板。
The acidic residues of the “acid–alcohol pair” in CYP51 enzymes are uniformly replaced with histidine. Herein, we adopt the Mycobacterium tuberculosis (mt) enzyme as a model system to investigate these residues’ roles in finely tuning the heme conformation, iron spin state, and formation and decay of the oxyferrous enzyme. Properties of the mtCYP51 and the T260A, T260V, and H259A mutants were interrogated using UV–Vis and resonance Raman spectroscopies. Evidence supports that these mutations induce comprehensive changes in the heme environment. The heme iron spin states are differentially sensitive to the binding of the substrate, dihydrolanosterol (DHL). DHL and clotrimazole perturb the local environments of the heme vinyl and propionate substituents. Molecular dynamics (MD) simulations of the DHL–enzyme complexes support that the observed perturbations are attributable to changes in the DHL binding mode. Furthermore, the rates of the oxyferrous formation were measured using stopped-flow methods. These studies demonstrate that both HT mutations and DHL modulate the rates of oxyferrous formation. Paradoxically, the binding rate to the H259A mutant–DHL complex was approximately four-fold that of mtCYP51, a phenomenon that is predicted to result from the creation of an additional diffusion channel from loss of the H259–E173 ion pair in the mutant. Oxyferrous enzyme auto-oxidation rates were relatively constant, with the exception of the T260V-DHL complex. MD simulations lead us to speculate that this behavior may be attributed to the distortion of the heme macrocycle by the substrate.
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DOI：
10.1016/s0006-291x(77)80131-9
发表时间：
1977-01-01
影响因子：
3.1
作者：
EISENSTEIN, L;DEBEY, P;DOUZOU, P
通讯作者：
DOUZOU, P
DOI：
10.1016/j.bbrc.2008.05.011
发表时间：
2008-07-25
影响因子：
3.1
作者：
Grinkova, Yelena V.;Denisov, Ilia G.;Sligar, Stephen G.
通讯作者：
Sligar, Stephen G.
DOI：
10.1073/pnas.96.16.8937
发表时间：
1999-08-03
影响因子：
11.1
作者：
Bellamine, A;Mangla, AT;Waterman, MR
通讯作者：
Waterman, MR
影响因子：
5.7
作者：
Cohen, J;Kim, K;Schulten, K
通讯作者：
Schulten, K
影响因子：
15
作者：
Hu, SZ;Smith, KM;Spiro, TG
通讯作者：
Spiro, TG