THEORY OF PHYSICAL PROPERTIES AND REACTIVITY IN P450

描述：这些持续计算的两个互补目标 代谢血红素蛋白普遍存在家族的研究， 细胞色素P450是：i）继续阐明最神秘的 所有P450同工酶共有的酶循环部分， 假定的瞬时物种;形成催化的途径 活性铁基（Fe=O）化合物I物质，来自两次还原的分子氧 ii）进一步表征的分子来源的 底物和产物特异性的酶循环部分， 通过比较结构功能研究， 细菌和哺乳动物脂肪酸羟化酶。 为了实现第一个目标，三种类型的计算研究是 计划好了 i）优化几何形状的计算和分析，以及 电子结构的瞬态血红素物种使用的技术， 从头计算量子化学直接评估他们参与 提出的途径，ii）计算推定的 瞬态物种使用半经验量子化学方法来帮助 实验学家试图通过光谱方法来识别它们， iii）进一步探索蛋白质在化合物I形成中的双重作用 即质子捐赠到远端氧和从它到 在最近的分子动力学模拟中确定了质子的最终来源 一种同工酶P450 eryF，通过使用相同的方法，a）扩展研究 与已知底物结合的另外两种P450同工酶P450 cam和P450 BM-3 结构和B）确定该途径的破坏是否可能是 已知特定突变效应的起源导致 功能障碍 从这些同工酶中预测的每一种同工酶的额外突变， 我们的研究导致功能障碍将建议我们的实验 合作者进行进一步评估。 第二个目标涉及细菌的比较结构功能研究， P450 BM-3和哺乳动物P450 4A 1和P4504 A11脂肪酸羟化酶 重点是如何在这些结合位点结构的差异， 同工酶可以改变其共同的底物和产物特异性， 底物，脂肪酸。 虽然这些细菌和哺乳动物同工酶具有 在优选的底物链长度上存在一些差异，它们具有显著的 不同的产品偏好; 4A 1和4A 11用于omega-1，BM-3用于omega-1， ω-2和ω-3区域选择性羟基化。 优选的 P4504 A11的脂肪酸底物的ω-羟基化代谢物是 直接涉及重要的生物功能，调节 血液流动和重要器官如肾脏的血管张力。 这些研究 需要使用所有三种同工酶的3D结构， 通过最近的实验和计算进展，特别是：i） 我们用X射线衍射法测定了P450-BM_3基质结构 合作者汤姆普洛斯，和ii）构建可靠的模型的能力， 4A 1和4A 11通过我们实验室最近开发和评估的方法 其他P450同工酶。 此外，我们的合作者Paul Ortiz de博士 蒙泰拉诺已同意制造所建议的4A 1和4A 11同工酶的突变体 通过我们提出的3D结构和结合位点，将进一步测试 模型的可靠性，以作出稳健的预测。

DESCRIPTION: The two complementary goals of these continuing computational studies of the ubiquitous family of metabolizing heme proteins the cytochrome P450s are: i) Continued elucidation of the most enigmatic portion of the enzymatic cycle common to all P450 isozymes involving putative transient species; the pathway to formation of the catalytically active, ferryl (Fe=O), Compound I species from the twice reduced dioxygen species (figure 2) ii) Further characterization of the molecular origin of substrate and product specificity in the portion of the enzymatic cycle that is unique for each P450 isozyme, by comparative structure function studies of bacterial and mammalian fatty acid hydroxylases. To accomplish the first goal, three types of computational studies are planned. i) Calculation and analysis of the optimized geometries and electronic structure of the transient heme species using the techniques of ab initio quantum chemistry to directly assess their involvement in the proposed pathway, ii) Calculation of the optical spectra of the putative transient species using semi-empirical quantum chemical method to aid experimentalists attempting to identify them by spectroscopic methods and iii) Further probing the dual role of the protein in Compound I formation namely proton donation to the distal oxygen and a link from it to an ultimate source of protons identified in recent molecular dynamic simulation of one isozyme P450eryF, by using the same methods to a) extend the studies to two other P450 isozymes, P450cam and P450BM-3 with known substrate bound structures and b) determine if disruption of this pathway could be the origin of the effect of specific mutations already known to lead to dysfunction. Additional mutations of each of these isozymes predicted from our studies to lead to dysfunction will be suggested to our experimental collaborators for further assessment. The second goal involves comparative structure function studies of bacterial P450BM-3 and mammalian P450 4A1 and P4504A11 fatty acid hydroxylases focusing on how differences in the binding site architecture of these isozymes can alter the substrate and product specificity of their common substrates, fatty acids. While these bacterial and mammalian isozymes have some differences in preferred substrate chain length, they have strikingly different product preferences; 4A1 and 4A11 for omega and BM-3 for omega-1, omega-2 and omega-3 regioselective hydroxylations. The preferred omega-hydroxylated metabolites of the fatty acid substrates of P4504A11 are directly implicated in important biological functions, the regulation of blood flow and vascular tone in vital organs such as kidney. These studies require use of 3D structures of all three isozymes and are now made possible by very recent experimental and computational advances, specifically: i) the X-Ray structure determination of a substrate bound P450-BM3 by our collaborator Tom Poulos, and ii) the ability to construct reliable models of 4A1 and 4A11 by methods recently developed and assessed in our laboratory for other P450 isozymes. In addition our collaborator Dr. Paul Ortiz de Montellano has agreed to make mutants of the 4A1 and 4A11 isozyme suggested by our proposed 3D structure and binding site that will further test the reliability of the models to make robust predictions.