Electronic/molecular structure of enzyme heme pockets

酶血红素口袋的电子/分子结构

• 批准号: 7028529
• 负责人: GERD N LA MAR
• 金额: $ 22.09万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7028529
• 关键词: active sites; chemical synthesis; cytochrome P450; enzyme structure; enzyme substrate; heme; heme oxygenase; horseradish peroxidase; lactoperoxidase; metalloenzyme; molecular shape; nuclear magnetic resonance spectroscopy; stereochemistry

DESCRIPTION (provided by applicant): We propose the detailed study of functionally relevant molecular/electronic structural and dynamic properties of a series of heme oxygenase, HO, enzymes and their complexes with substrate/reaction intermediates in variable oxidation/spin/liagtion states, using high resolution solution 2D/3D NMR. HO, found in vertebrates, plants and bacteria, acts by a common mechanism and set of intermediates, using heme as both substrate and cofactor, to stereoselectively cleave heme into a-biliverdin, iron and CO. HO is unique in using the hydroperoxy species as its activated form, and the structural properties of the active site that stabilize the species are not well understood except that ordered water molecules within a distal H-bond network are involved. We select 3 HOs, isozyme #1 from human, hHO, and those from 2 pathogenic bacteria C. diphtheriae (CofHO) and N. meningitidis (NmHO), which share a common fold, but exhibit variable sequence homology for the residues involved in the H-bonding network. The target derivatives are substrate-free or app-HO, resting state HO-hemin-H/jO, HO-hemin-CN as a model for the unstable oxy complex, and HO-hemin-OH as a model for the reactive hydroperpxy species. Since all but 1 targeted HO derivative are paramagnetic, emphasis is placed on utilizing appropriately tailored 1D/2D/3D NMR to extract the wealth of unique information in hyperfine shifts. We will develop a new and highly sensitive NMR probe that directly reflect the degree of H-bonding between axial ligand to the hemin and the distal ordered-water/H-bond network, using the pair of complex HO-hemin-H2O/-OH, and to use this probe, as well as previously established procedures, to provide a detailed characterization of the solution structure. Our interests focus on comparison of local solution with cryogenic crystallographic molecular structure, with particular attention paid to extended H-bond networks with some remarkably robust H-bonds, and the ordered water molecules within these networks. We emphasize comparative studies among the various derivatives of 1 HO, and among the different HOs for a given derivative, to elucidate the relationship between variable strength H-bonds and axial ligand properties. In addition, we will characterize the influence of HO, substrate or intermediates and their axial ligands on dynamic properties related to entry and exit of substrate. Lastly, for NmHO, we will characterize the influence of heme substituents on its seating in the active site, determine the structure of the crystallographically disordered C-terminus found folded into the active site in solution, and illuminate the role of the C-terminus and the unique active site Cys113 in multiple, functionally relevant, microheterogeneities. The detailed description of the molecular structural and dynamic properties of heme oxygenase will improve our understanding of the varied roles of mammalian enzymes. The elucidation of the similarities and differences between bacterial and mammalian heme oxygenase will improve prospects for the design of selective inhibitors for the enzyme in pathogenic bacteria.

描述（由申请人提供）：我们建议使用高分辨率溶液 2D/3D NMR，对一系列血红素加氧酶、H2O、酶及其与可变氧化/自旋/连接状态下的底物/反应中间体的复合物的功能相关的分子/电子结构和动态特性进行详细研究。 H2O 存在于脊椎动物、植物和细菌中，通过共同的机制和一组中间体发挥作用，使用血红素作为底物和辅助因子，将血红素立体选择性地裂解为 α-胆绿素、铁和 CO。 H2O 的独特之处在于使用氢过氧物质作为其活化形式，并且稳定该物质的活性位点的结构特性尚不清楚，除了在 涉及远端氢键网络。我们选择了 3 个 HO，即来自人类 hHO 的同工酶 #1，以及来自 2 种病原菌白喉杆菌 (CofHO) 和脑膜炎奈瑟菌 (NmHO) 的同工酶，它们具有共同的折叠，但在氢键网络中涉及的残基表现出不同的序列同源性。目标衍生物是无底物或 app-H2O、静息态 HO-血红素-H/jO、HO-血红素-CN 作为不稳定氧复合物的模型，以及 HO-血红素-OH 作为活性水过氧化物的模型。由于除 1 种目标 H2O 衍生物之外的所有衍生物都是顺磁性的，因此重点放在利用适当定制的 1D/2D/3D NMR 来提取超精细位移中丰富的独特信息。我们将开发一种新型高灵敏度核磁共振探针，使用一对复杂的HO-血红素-H2O/-OH，直接反映轴向配体与血红素和远端有序水/氢键网络之间的氢键结合程度，并使用该探针以及先前建立的程序来提供溶液结构的详细表征。我们的兴趣集中在局部溶液与低温晶体分子结构的比较，特别关注具有一些非常坚固的氢键的扩展氢键网络，以及这些网络内的有序水分子。我们强调 1 H2O 的各种衍生物之间以及给定衍生物的不同 H2O 之间的比较研究，以阐明不同强度的氢键和轴向配体性质之间的关系。此外，我们将表征 H2O、底物或中间体及其轴向配体对与底物进入和退出相关的动态特性的影响。最后，对于 NmHO，我们将表征血红素取代基对其在活性位点中的位置的影响，确定在溶液中折叠到活性位点中的晶体学无序 C 末端的结构，并阐明 C 末端和独特的活性位点 Cys113 在多个功能相关的微观异质性中的作用。对血红素加氧酶的分子结构和动态特性的详细描述将提高我们对哺乳动物酶的不同作用的理解。阐明细菌和哺乳动物血红素加氧酶之间的相似性和差异将改善病原菌中该酶的选择性抑制剂的设计前景。