THEORY OF PHYSICAL PROPERTIES AND REACTIVITY IN P450

P450 的物理性质和反应性理论

• 批准号: 2370984
• 负责人: GILDA H LOEW
• 金额: $ 22.72万
• 依托单位: MOLECULAR RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2370984
• 关键词: X ray crystallography; bacterial proteins; chemical binding; computer simulation; conformation; cytochrome P450; enzyme substrate complex; fatty acid metabolism; fatty acylation; hemoprotein structure; hydroxy fatty acid; isozymes; molecular dynamics; molecular site; nuclear magnetic resonance spectroscopy; oxygenases; physical model; physical property; protein structure function; quantum chemistry

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.

描述：这些持续计算的两个互补目标 无处不在的代谢血红素蛋白家族的研究 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，包括 假定的暂态物种；催化形成的途径 活性铁基(Fe=O)，化合物I来自二次还原的氧气 物种(图2)II)进一步刻画分子起源 酶循环中底物和产物的专一性 通过比较结构功能研究，每个P450同工酶都是独一无二的 细菌和哺乳动物的脂肪酸羟基酶。 为了实现第一个目标，有三种类型的计算研究 有计划的。I)计算和分析优化的几何形状和 瞬时亚铁血红素物种的电子结构研究 从头算量子化学，直接评估他们参与的 提出的途径，II)推定的光谱的计算 用半经验量子化学方法辅助瞬变物种 实验者试图通过光谱方法来识别它们 三)进一步探讨蛋白质在化合物I形成中的双重作用 即质子对远端氧的贡献和从它到 最新分子动力学模拟确定的质子终极来源 一种同工酶P450eryF，通过使用相同的方法a)扩展研究 与另两种已知底物结合的P450同工酶P450cam和P450BM-3 结构和b)确定这一途径的中断是否可能是 已知的特定突变的影响的起源 功能障碍。这些同工酶中的每一种预测的额外突变 我们导致功能障碍的研究将被建议给我们的实验 合作者进行进一步评估。 第二个目标涉及细菌的比较结构和功能研究。 P450BM-3与哺乳动物P450 4A1和P4504A11脂肪酸羟基酶 重点关注这些结合位点体系结构的差异 同工酶可以改变它们共同的底物和产物的特异性 底物，脂肪酸。而这些细菌和哺乳动物的同工酶 在优选底物链长上有一些差异，它们具有显著的 不同的产品偏好；omega的4A1和4A11，omega-1的BM-3， Omega-2和omega-3区域选择性羟基化。首选的 P4504A11脂肪酸底物的欧米伽羟化代谢产物为 直接牵涉到重要的生物功能，调节 肾脏等重要器官的血流和血管紧张度。这些研究 需要使用所有三种同工酶的3D结构，现在可以 通过最新的实验和计算进展，具体地说：i) OUR法测定底物结合的P450-BM3的X射线结构 合作者Tom Poulos，以及ii)构建可靠模型的能力 4A1和4A11是我们实验室最新开发和评估的方法 其他P450同工酶。此外，我们的合作者保罗·奥尔蒂斯·德博士 蒙特拉诺已同意对所建议的4A1和4A11同工酶进行突变 通过我们建议的3D结构和结合部位，将进一步测试 模型的可靠性，以做出稳健的预测。