Hernandez Cynthia E, Kumar Santosh, Liu Hong, Halpert James R
Department of Pharmacology and Toxicology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1031, USA.
Arch Biochem Biophys. 2006 Nov 1;455(1):61-7. doi: 10.1016/j.abb.2006.08.024. Epub 2006 Sep 25.
Based on the X-ray crystal structures of 4-(4-chlorophenyl)imidazole (4-CPI)- and bifonazole (BIF)-bound P450 2B4, eight active site mutants at six positions were created in an N-terminal modified construct termed 2B4dH and characterized for enzyme inhibition and catalysis. I363A showed a >4-fold decrease in differential inhibition by BIF and 4-CPI (IC(50,BIF)/IC(50,4-CPI)). F296A, T302A, I363A, V367A, and V477A showed a 2-fold decreased k(cat) for 7-ethoxy-4-trifluoromethylcoumarin O-deethylation, whereas V367A and V477F showed an altered K(m). T302A, V367L, and V477A showed >4-fold decrease in total testosterone hydroxylation, whereas I363A, V367A, and V477F showed altered stereo- and regioselectivity. Interestingly, I363A showed a 150-fold enhanced k(cat)/K(m) with testosterone, and yielded a new metabolite. Furthermore, testosterone docking into three-dimensional models of selected mutants based on the 4-CPI-bound structure suggested a re-positioning of residues 363 and 477 to yield products. In conclusion, our results suggest that the 4-CPI-bound 2B4dH/H226Y crystal structure is an appropriate model for predicting enzyme catalysis.
基于与4-（4-氯苯基）咪唑（4-CPI）和联苯苄唑（BIF）结合的细胞色素P450 2B4的X射线晶体结构，在一种称为2B4dH的N端修饰构建体中，在六个位置创建了八个活性位点突变体，并对其酶抑制和催化特性进行了表征。I363A对BIF和4-CPI的差异抑制作用（IC(50,BIF)/IC(50,4-CPI)）降低了4倍以上。F296A、T302A、I363A、V367A和V477A对7-乙氧基-4-三氟甲基香豆素O-脱乙基反应的催化常数（k(cat)）降低了2倍，而V367A和V477F的米氏常数（K(m)）发生了改变。T302A、V367L和V477A对总睾酮羟化反应的活性降低了4倍以上，而I363A、V367A和V477F的立体和区域选择性发生了改变。有趣的是，I363A对睾酮的催化常数与米氏常数之比（k(cat)/K(m)）提高了150倍，并产生了一种新的代谢产物。此外，基于与4-CPI结合的结构将睾酮对接至所选突变体的三维模型表明，残基363和477重新定位以产生产物。总之，我们的结果表明，与4-CPI结合的2B4dH/H226Y晶体结构是预测酶催化作用的合适模型。
