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

细胞色素P450(P450)是含血红素的单加氧酶超家族之一，在生物体中催化多种有机底物的生物转化反应。根据水溶性P450的X-射线晶体结构和P450的氨基酸序列，人们对P450的结构与功能关系的认识取得了很大的进展。结合这一知识和定点突变技术，从血红素的掺入(生物化学，27，b4138-4141(1988))和推测的远端部位的结构(生物化学，28,6848-6857(1989)；生物化学，30,1490-1496(1991)；生物化学，30,4659-4662(1991))，阐明了膜结合的P450_d(CYP1A2)的结构。在这项研究中，我们发现了P450_d远端的Glu318(CyP1A2)在P450_d分子远端的堆积或构象稳定性中的重要作用(生物化学，30,11206-11211(1991年))，以及Glu318在P450_d的催化功能中的重要作用(生物化学，31,1528-1531(1992年))。此外，我们还发现P450_d(CYP1A2)的Lys94、Lys99、LYS105、Lys440、Lys453、Arg455、Lys463和Arg簇Arg135-Arg136-Arg137通过在P450_d和NADPH-P450还原酶之间形成离子桥和/或通过在两个蛋白质(J.Biol)之间的界面上定向电子转移而参与了分子间电子转移过程。化学，266,3372-3375(1991))。P450_d的318位氨基酸(P450的Glu或Asp；P450_lt；CaM和gt；的Asp251)比Thr(P450_d的Thr319；P450_lt；CaM和gt；的Thr252)保守的极性氨基酸对P450结合的氧分子的激活有显著的贡献。

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.