Studies on the Redox Conformational Changes and Substrate Adaptability Changes in Cytochrome P-450_d Mutants

细胞色素P-450_d突变体氧化还原构象变化及底物适应性变化的研究

• 批准号: 02044017
• 负责人: HATANO Masahiro
• 金额: $ 3.52万
• 依托单位: Tohoku University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for international Scientific Research
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02044017/
• 关键词: Cytochrome P450; Membrane-bound protein; Structure-function relationship of enzyme; Electron transfer in proteins; Stabilization of helices; Motional fluctuation of the amino acid side chains; X-ray crystal structure analysis; Conformational change

The cytochrome P450(P450) is one of the superfamily of heme-containing monooxygenases which catalyze many types of biotransformation reactions of organic substrates in living organisms. Significant progress in understanding the P450 structure-function relationship has been made on the basis of the X-ray crystal structure of water-soluble P450_<cam> and alignments of amino acids of P450s. By combining this knowledge and site-directed mutagenesis technique, the structure of membrane-bound P450_d(CYP1A2) was elucidated with respect to the heme incorporation (Biochemistry, 27, b4138-4141(1988)) and the structure of putative distal site (Biochemistry, 28, 6848-6857 (1989) ; Biochemistry, 30, 1490-1496(1991) ; Biochbmistry, 30, 4659-4662 (1991)). In this project we found the important role of Glu318 at the putative distal site' of P450_d(CYP1A2) in the packing or the conformational stability of the putative distal site of the P450_d molecule (Biochemistry, 30, 11206-11211(1991)) and the important role of the Glu318 in the catalytic function of the P450_d (Biochbmistry, 31, 1528-1531(1992)). Furthermore, we found that Lys94, Lys99, LYS105, Lys440, Lys453, Arg455, Lys463, and the Arg cluster, Arg135-Arg136-Arg137, of P450_d(CYP1A2) participate in the inter-molecular electron transfer process by forming ionic bridges between the P450_d and NADPH-P450 reductase and/or by orienting appropriate geometry for electron transfer on the interfacial surface between the two proteins (J. Biol. Chem., 266, 3372-3375(1991)). Well-conserved polar amino acids at position 318 of P450_d (Glu or Asp of P450s ; Asp251 of P450_<cam>) prior to the conserved Thr (Thr3l9 for P450_d ; Thr252 for P450_<cam>) significantly contribute to the activation of the oxygen molecule bound to P450.

细胞色素P450（Cytochrome P450，P450）是含血红素单加氧酶超家族中的一员，在生物体中催化多种有机底物的生物转化反应。基于水溶性P450的X-射线晶体结构和氨基酸序列比对，对P450结构-功能关系的认识取得了重大进展<cam>。结合这一知识和定点突变技术，阐明了膜结合P450_d（CYP 1A 2）的结构与血红素掺入的关系（Biochemistry，27，b4138-4141（1988））和推定的远端位点的结构（Biochemistry，28，6848-6857（1989）; Biochemistry，30，1490-1496（1991）; Biochbmistry，30，4659-4662（1991））。本研究发现，位于P450_d（CYP 1A 2）远端位点的Glu 318在P450_d分子的构象稳定性中起重要作用（Biochemistry，30，11206-11211（1991））和Glu 318在P450_d的催化功能中的重要作用。（Biochbmistry，31，1528-1531（1992））。此外，我们发现Lys 94，Lys 99，LYS 105，Lys 440，Lys 453，Arg 455，Lys 463，以及Arg簇，Arg 135-Arg 136-Arg 137，P450_d（CYP 1A 2）通过在P450_d和NADPH-P450还原酶之间形成离子桥参与分子间电子转移过程，和/或或通过在两种蛋白质之间的界面表面上定向用于电子转移的适当几何形状（J. Biol. Chem.，266，3372-3375（1991））。在保守的Thr（对于P450_d为Thr 319;对于P450_为Thr 252）之前的P450_d的位置318处的高度保守的极性氨基酸（P450_的Glu或Asp; P450_的Asp <cam>251<cam>）显著地促进与P450结合的氧分子的活化。

项目成果

A. G. Krainev, T. Shimizu, M. Ishigooka, K. Hiroya, M. Hatano, and Y. Fujii-Kuriyama: "Absorption Spectral Study of Cytochrome P450_d-Phenyl Isocyanide Complexes Effects of Mutations at the Putative Distal Site on the Conformational Stability." Biochemist

A. G. Krainev、T. Shimizu、M. Ishigooka、K. Hiroya、M. Hatano 和 Y. Fujii-Kuriyama：“细胞色素 P450_d-苯基异氰化物复合物的吸收光谱研究假定远端位点突变对构象稳定性的影响。

T.Shimizu,A.J.Md Sadeque,G.N.Sadeque,M.Hatano et al.: "Ligand Binding Studied of Engineered Cytochreme pー450_d wild Type,proximal Mutants and distal Mutants" Biochemistry. 30. 1491-1496 (1991)

T.Shimizu、A.J.Md Sadeque、G.N.Sadeque、M.Hatano 等人：“工程细胞色素 pー450_d 野生型、近端突变体和远端突变体的配体结合研究”生物化学 30. 1491-1496 (1991)。

H.Furuya,T.Shimizu,K.Hirano,M.Hatano,and Y.Fujiiーkuriyama et al.: "SiteーDirected Mutageneses of Rat Liver Cytochrome pー450_d:Catalytic Activities toward Benzphetamine and 7ーEthoxycoumarin" Biochemistry. 28. 6848-6857 (1989)

H.Furuya、T.Shimizu、K.Hirano、M.Hatano 和 Y.Fujii-kuriyama 等人：“大鼠肝脏细胞色素 p-450_d 的定点诱变：对苯异丙胺和 7-乙氧基香豆素的催化活性”生物化学。 28. 6848-6857 (1989)

H.Furuya,T.Shimizu,M.Hatano and Y.FujiiーKuriyama: "Mutations at the Distal,and proximal Sites of Cytochrome Pー450_d Changed RegioーSelectivity of Acetanilide Hydroxylations" Biochemical and Biophysical Research Communications. 160. 669-676 (1989)

H.Furuya、T.Shimizu、M.Hatano 和 Y.Fujii Kuriyama：“细胞色素 P-450_d 远端和近端位点的突变改变了乙酰苯胺羟基化的区域选择性”生物化学和生物物理研究通讯。 (1989)

T.Shimizu,T.Tateishi M.Hatano,and Y.FujiiーKuriyama: "Probing the Role of Lysines and Arginines in the Catalytic Function of Cytochrome pー450_d by SiteーDirected Mutagenesis" J.Biol.Chem.266. (1991)

T.Shimizu、T.Tateishi M.Hatano 和 Y.Fujii Kuriyama：“通过定点诱变探讨赖氨酸和精氨酸在细胞色素 p450_d 催化功能中的作用”J.Biol.Chem.266。