Study of substrate specificity of human aromatase by site directed mutagenesis

被引:24
作者
Auvray, P
Nativelle, C
Bureau, R
Dallemagne, P
Séralini, GE
Sourdaine, P
机构
[1] Univ Caen, IBBA, Lab Biochim & Biol Mol, F-14032 Caen, France
[2] CERMN, Lab Pharmacochim, Caen, France
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 2002年 / 269卷 / 05期
关键词
aromatase; site-directed mutagenesis; molecular modeling; androgens; inhibitors;
D O I
10.1046/j.1432-1033.2002.02779.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Human aromatase is responsible for estrogen biosynthesis and is implicated, in particular, in reproduction and estrogen-dependent tumor proliferation. The molecular structure model is largely derived from the X-ray structure of bacterial cytochromes sharing only 15-20% identities with hP-450arom. In the present study, site directed mutagenesis experiments were performed to examine the role of K119, C124, I125, K130, E302, F320, D309, H475, D476, S470, I471 and I474 of aromatase in catalysis and for substrate binding. The catalytic properties of mutants, transfected in 293 cells, were evaluated using androstenedione, testosterone or nor-testosterone as substrates. In addition, inhibition profiles for these mutants with indane or indolizinone derivatives were obtained. Our results, together with computer modeling, show that catalytic properties of mutants vary in accordance with the substrate used, suggesting possible differences in substrates positioning within the active site. In this respect, importance of residues H475, D476 and K130 was discussed. These results allow us to hypothesize that E302 could be involved in the aromatization mechanism with nor-androgens, whereas D309 remains involved in androgen aromatization. This study highlights the flexibility of the substrate-enzyme complex conformation, and thus sheds new light on residues that may be responsible for substrate specificity between species or aromatase isoforms.
引用
收藏
页码:1393 / 1405
页数:13
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