细胞色素P450酶电化学生物传感器的构建及其药物代谢应用

药物代谢过程是药物在体内产生药效和毒性的主要过程,发展廉价、方便、快速、高通量的体外药物代谢研究方法对新药的开发和设计、给药的方法和剂量、临床药物的检测等都有重要的指导意义. 细胞色素P450酶（CYP450酶）在药物的I相反应中起到关键作用,以电极代替辅酶NADPH提供CYP450酶催化反应过程中需要的两个电子,构建CYP450酶电化学生物传感器可实现药物的初步筛选. 大量研究表明,CYP450酶在电极表面合适的固定方法与电极材料可有效提高传感器的检测性能. 本文主要综述近年来CYP450酶电化学生物传感器的构建及其在药物代谢研究方面的应用,并展望其研发前景.     

细胞色素C的薄层光谱电化学研究

细胞色素C(Cyt. C)在电极界面上的电子传递十分缓慢,只有在适当的电子迁移中介体(Mediator)或促进剂(Promoter)参与下才能以较快的速度进行反应,本文报道了以4,4′-二硫基联吡啶(PySSPy)作电极反应促进剂,用薄层光谱电化学技术研究细胞色素C在金微网栅薄层透光电极界面上的电化学过程,测定了电极反应的热力学参数E~(o′)及n,并与循环伏     

亚甲蓝修饰电极推动的血红素蛋白质直接电子转移反应

本文研究了几种血红素蛋白质包括牛血红蛋白, 人肌红蛋白和马心细胞色素C在亚甲蓝修饰电极上的非均相电子转移反应, 采用光透薄层光谱电化学法监测了血红素蛋白的直接电化学反应过程, 并进行了动力学研究。     

磷灰石通道离子替换对细胞色素C直接电化学的影响

采用沉淀法合成了以OH^-、F^-、Cl^-为通道离子的磷灰石纳米晶体。利用其独特的多位点吸附特性,研究了磷灰石不同通道离子替换对细胞色素c直接电化学的影响。在pH7．0的磷酸盐缓冲溶液中,细胞色素C在磷灰石修饰玻碳电极表面于0．074V(us．Ag／AgCl)附近有一对准可逆的氧化还原峰,为细胞色素C血红素辅基Fe(Ⅲ）／Fe(Ⅱ)电对的特征峰。细胞色素C与磷灰石之间的静电作用使深藏在细胞色素C内部的电活性中心靠近电极表面,加速了细胞色素C在玻碳电极表面扩散控制的准可逆单电子转移过程。其中氟磷灰石对细胞色素C直接电化学的促进作用最显著,羟基磷灰石次之,氯磷灰石最弱。     

细胞色素p450的结构与催化机理

细胞色素P450酶是广泛存在的含亚铁血红素单加氧酶, 参与甾类激素的合成、脂溶性维生素代谢、多不饱和脂肪酸转换为生物活性分子, 以及致癌作用和药物代谢. 综述了细胞色素p450结构与功能的关系, 特别是细胞色素P450活性位点经历大幅度开/关运动结合底物和释放产物以及电子迁移途径.     

细胞色素c在羟基磷灰石修饰玻碳电极上的直接电化学

采用沉淀法合成羟基磷灰石纳米晶体，由于具有独特的多吸附位点特征，羟基磷灰石可作为一种新型电子传递促进剂用于细胞色素c的直接电化学研究．在pH7．0的磷酸盐缓冲溶液中，细胞色素c在羟基磷灰石修饰玻碳电极表面于0．074V(vs．Ag/AgC1)处有一对准可逆的氧化还原峰，为细胞色素c血红素辅基Fe(Ⅲ)／Fe(Ⅱ)电对的特征峰．实验结果表明细胞色素c与羟基磷灰石之间的静电作用．促进了细胞色素c在玻碳电极表面扩散控制的准可逆单电子转移过程．讨论了电位扫描速度、溶液离子强度对细胞色素c直接电化学的影响．     

细胞色素C551在ITO电极上的直接电化学

在不加任何电子转移促进剂的条件下,用循环伏安法(CV)和微分脉冲伏安法(DPV)观察到细胞色素C551在ITO导电玻璃上的直接电化学行为.结果显示电极反应过程为准可逆性质,计算得到细胞色素C551的扩散系数、式电位和异相电子转移标准速率常数,并对其在ITO导电玻璃电极上的电子转移机制进行了初步分析.     

细胞色素C在单糖修饰金电极上的直接电化学

Hill等发现在4,4′-联吡啶存在时,细胞色素C在金电极上能进行准可逆的电化学反应。在研究细胞色素C的直接电化学过程中,人们又发现一些生物小分子如氨基酸、嘌呤等对细胞色素C的电化学反应有促进作用,但迄今未见有关糖类分子对细胞色素C电化学反应促进作用研究的报道。本文研究了5种单糖对细胞色素C电化学反应的促进作用。     

吡啶、2-甲基吡啶存在下细胞色素c碱式异构化和配体细胞色素c配合物的电化学研究

用半胱氨酸修饰的金电极研究了吡啶、2 甲基吡啶存在下细胞色素c碱式异构化和配体结合细胞色素c的电化学。在此电极上 ,细胞色素c可发生准可逆的电极反应而吡啶结合细胞色素c和 2 甲基吡啶结合细胞色素c在循环伏安图上只给出还原峰。高浓度 (1.2 7mol·L- 1)的吡啶和 2 甲基吡啶可诱导碱式细胞色素c在中性条件下生成。进一步的研究表明 ,这种诱导作用与配体和细胞色素c的键合无关     

双功能有机物修饰电极的修饰层结构对催化细胞色素C的电极反应的影响

用光谱电化学法研究了双功能有机物健那绿的电化学性质。在铂电极上,健那绿发生四步单电子传递过程。在健那绿修饰电极上细胞色素C的直接、非均相电子传递反应是准可逆氧化还原反应,电极反应速率常数K_s=4.17×10~(-4)cm/s。电极表面的修饰方法不同对细胞色素C的电极反应有很大影响。     

The Cytochrome P450 Engineering Database: integration of biochemical properties

Background  

Cytochrome P450 monooxygenases (CYPs) form a vast and diverse enzyme class of particular interest in drug development and a high biotechnological potential. Although very diverse in sequence, they share a common structural fold. For the comprehensive and systematic comparison of protein sequences and structures the Cytochrome P450 Engineering Database (CYPED) was established. It was built up based on an extensible data model that enables its functions readily enhanced.     

Pharmacophore modeling and in silico screening for new P450 19 (aromatase) inhibitors

Cytochrome P450 19 (P450 19, aromatase) constitutes a successful target for the treatment of breast cancer. This study analyzes chemical features common to P450 19 inhibitors to develop ligand-based, selective pharmacophore models for this enzyme. The HipHop and HypoRefine algorithms implemented in the Catalyst software package were employed to create both common feature and quantitative models. The common feature model for P450 19 includes two ring aromatic features in its core and two hydrogen bond acceptors at the ends. The models were used as database search queries to identify active compounds from the NCI database.     

单核锰配合物对几类氧化-还原酶的功能模拟

金属锰存在于多种氧化-还原酶中。其中,锰超氧化物歧化酶(Mn Superoxide Dismutase)及锰过氧化物酶(Mn Peroxidase)活性中心的锰结合位点,根据其结构特征,可将其视为精巧的单核锰配合物,因而人们通常以小分子配合物来模拟酶活性中心的结构及功能;另外,细胞色素P450是一种能够催化烯烃环氧化等诸多反应的单加氧酶,某些单核锰配合物能够起到与其类似的催化氧化效果。本文介绍了几类氧化-还原酶的结构特征和催化机理,总结了以小分子锰配合物作为酶功能模拟物的研究进展。     

Engineering and assaying of cytochrome P450 biocatalysts

Cytochrome P450s constitute a highly fascinating superfamily of enzymes which catalyze a broad range of reactions. They are essential for drug metabolism and promise industrial applications in biotechnology and biosensing. The constant search for cytochrome P450 enzymes with enhanced catalytic performances has generated a large body of research. This review will concentrate on two key aspects related to the identification and improvement of cytochrome P450 biocatalysts, namely the engineering and assaying of these enzymes. To this end, recent advances in cytochrome P450 development are reported and commonly used screening methods are surveyed.     

高通量筛选策略在细胞色素P450单加氧酶定向进化中的应用

细胞色素P450单加氧酶具有催化活性混杂性的特点,可以催化多种氧化反应,因而在生物催化领域受到了极大的关注。然而P450单加氧酶往往存在催化活性低、稳定性差、区域和立体选择性不理想等问题,从而限制了其在生物催化领域的广泛运用。蛋白质定向进化的发展与运用为改善P450单加氧酶的催化性能提供了有效的途径,而一种高效的高通量筛选策略是保证酶蛋白定向进化成功实施的关键。本文综述了P450单加氧酶定向进化过程中高通量筛选策略的最新进展。     

WhichP450: a multi-class categorical model to predict the major metabolising CYP450 isoform for a compound

In the development of novel pharmaceuticals, the knowledge of how many, and which, Cytochrome P450 isoforms are involved in the phase I metabolism of a compound is important. Potential problems can arise if a compound is metabolised predominantly by a single isoform in terms of drug–drug interactions or genetic polymorphisms that would lead to variations in exposure in the general population. Combined with models of regioselectivities of metabolism by each isoform, such a model would also aid in the prediction of the metabolites likely to be formed by P450-mediated metabolism. We describe the generation of a multi-class random forest model to predict which, out of a list of the seven leading Cytochrome P450 isoforms, would be the major metabolising isoforms for a novel compound. The model has a 76% success rate with a top-1 criterion and an 88% success rate for a top-2 criterion and shows significant enrichment over randomised models.     

Electrochemical study of the interaction between cytochrome P450sccK201E and cholesterol

Cytochrome P450sccK201E, mutated form of cytochrome P450scc native recombinant (P450sccNR), was employed to study the enzyme–substrate interaction. The detection of the cholesterol was performed by electrochemical method using cyclic voltammetry (CV) and chronoamperometry measurements. The biochemical analysis was realized to observe the electrochemical responses of the engineerized enzyme to three different forms of cholesterol: free, low-density lipoprotein (LDL) and high-density lipoproteins (HDL). Compared to cytochrome P450sccNR, the cytochrome P450sccK201E displays a different behavior in the interaction with the substrate detection.

The results show that the engineerized enzyme can be utilized for the cholesterol detection in biosensor field.     

In Silico Analysis of PORD Mutations on the 3D Structure of P450 Oxidoreductase

Cytochrome P450 oxidoreductase (POR) is a membrane-bound flavoprotein that helps in transferring electrons from its NADPH domain to all cytochrome P450 (CYP450) enzymes. Mutations in the POR gene could severely affect the metabolism of steroid hormones and the development of skeletal muscles, a condition known as Cytochrome P450 oxidoreductase deficiency (PORD). PORD is associated with clinical presentations of disorders of sex development, Antley and Bixler’s syndrome (ABS), as well as an abnormal steroid hormone profile. We have performed an in silico analysis of POR 3D X-ray protein crystal structure to study the effects of reported mutations on the POR enzyme structure. A total of 32 missense mutations were identified, from 170 PORD patients, and mapped on the 3D crystal structure of the POR enzyme. In addition, five of the missense mutations (R457H, A287P, D210G, Y181D and Y607C) were further selected for an in-depth in silico analysis to correlate the observed changes in POR protein structure with the clinical phenotypes observed in PORD patients. Overall, missense mutations found in the binding sites of POR cofactors could lead to a severe form of PORD, emphasizing the importance of POR cofactor binding domains in transferring electrons to the CYP450 enzyme family.     

Cytochrome p450 compound I

Cytochrome P450 enzymes (P450s) comprise a large class of enzymes that effect numerous oxidations in nature. The active oxidants in P450s are thought to be iron(IV)-oxo porphyrin radical cations termed Compounds I, and these intermediates have been sought since the discovery of P450s 40 years ago. We report formation of the Compound I derivative of a P450 enzyme by laser flash photolysis oxidation of the corresponding Compound II species, an iron(IV)-oxo neutral porphyrin intermediate. The Compound II derivative in turn was produced by oxidation of the P450 with peroxynitrite, which effected a net one-electron, oxo-transfer reaction to the iron(III) atom of the resting enzyme. For the P450 studied in this work, CYP119 from the thermophile Sulfolobus solfactaricus, the P450 Compound II derivative was stable for seconds at ambient temperature, and the Compound I transient decayed with a lifetime of ca. 200 ms.     

Enantioselective epoxidation of terminal alkenes to (R)- and (S)-epoxides by engineered cytochromes P450 BM-3

Cytochrome P450 BM-3 from Bacillus megaterium was engineered for enantioselective epoxidation of simple terminal alkenes. Screening saturation mutagenesis libraries, in which mutations were introduced in the active site of an engineered P450, followed by recombination of beneficial mutations generated two P450 BM-3 variants that convert a range of terminal alkenes to either (R)- or (S)-epoxide (up to 83 % ee) with high catalytic turnovers (up to 1370) and high epoxidation selectivities (up to 95 %). A biocatalytic system using E. coli lysates containing P450 variants as the epoxidation catalysts and in vitro NADPH regeneration by the alcohol dehydrogenase from Thermoanaerobium brockii generates each of the epoxide enantiomers, without additional cofactor.