Electronic/Molecular Structure of Enzyme Heme Pockets

酶血红素袋的电子/分子结构

• 批准号: 6741431
• 负责人: GERD N LA MAR
• 金额: $ 14.7万
• 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 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6741431
• 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) This proposal deals with experimental approaches to quantitative characterization of both the molecular and electronic structures of the active sites of moderate-sized (30-80 kDa) heme-containing enzymes in order to understand the mechanism (peroxidase vs. oxygenase) and stereospecificity (alpha vs. beta, gamma, or delta-meso heme cleavage) of the reactions with substrate. Solution NMR spectroscopy of the active site of heme enzymes in paramagnetic states is ideally suited for providing detailed description of such active sites, where advantage is taken of both the increased spectral dispersion that significantly facilitates resolution for active site residues, and the great wealth of novel structural information contained in the hyperfine shifts responsible for the shift dispersion. Methodological approaches will emphasize more quantitative structural studies (internuclear distance) of 30-40 kDa low-spin, ferric enzymes and extension of both assignments and structural analyses to larger 78 kDa) low-spin, ferric, and more strongly paramagnetic, 30-40 kDa high-spin, ferric heme enzymes. The methodological advances are expected to contribute not only to effective structural characterization of our own target enzymes, but to contribute significantly to the NMR investigation of a large variety of other paramagnetic metalloproteins currently pursued in other research laboratories. The optimally developed NMR strategies will be applied to determining in detail the effect of mutation and ligation on the nature of the heme cavity and substrate binding pocket of 44 kDa horseradish peroxidase, and elucidating the heme electronic structure that results from the covalent heme links to the protein matrix in 78 kDa lactoperoxidase. Studies on ligated and substrate-bound human heme oxygenase will focus on reconciling the apparent structural differences between the ligated complex in solution and the unligated complex in the crystal with respect to the position of the distal helix, the nature of the distal hydrogen-bond donor to the bound ligand, the identity of the titrating group that inactivates the protein, the exact origin of steric tilt of the ligand in the direction of the alpha-meso position, and the nature of the distal hydrogen-bonds between two Tyr that are likely involved in "closing" the substrate cavity prior to reaction. The effect of mutation on these structural features will also be examined. The prospects for definitive solution NMR studies of cytochrome P450 will also be explored. It is expected that a considerable improved understanding of the solution structures of the target enzyme and the relationship to their functions will result from these studies.

描述：（由申请人提供）本提案涉及实验 定量表征分子和 中等大小（30-80 kDa）的活性位点的电子结构 含血红素的酶，以了解机制（过氧化物酶与 加氧酶）和立体特异性（α相对于β、γ或δ-内消旋血红素 裂解）与底物的反应。的溶液NMR光谱 顺磁状态的血红素酶的活性位点理想地适合于 提供了对这些活性位点的详细描述， 增加的光谱色散， 活性位点残基的解析，以及大量的新结构 超精细位移中包含的信息 分散体方法论将强调更多的量化 30-40 kDa低自旋三价铁的结构研究（核间距） 酶和扩展的分配和结构分析，以更大的78 kDa）低自旋，铁，和更强的顺磁性，30-40 kDa高自旋， 铁血红素酶 预计方法上的进步不仅有助于有效地 我们自己的目标酶的结构特征，但有助于 显着的核磁共振研究了大量的各种其他顺磁 金属蛋白目前在其他研究实验室进行。 最佳开发的NMR策略将被应用于详细确定 突变和连接对血红素腔性质的影响， 44 kDa辣根过氧化物酶的底物结合口袋，并阐明了 血红素电子结构，由共价血红素连接到 78 kDa乳过氧化物酶中的蛋白质基质。研究了连接和 与底物结合的人类血红素加氧酶将专注于调和明显的 溶液中连接的复合物与 晶体中未连接的复合物相对于远端的位置 螺旋，结合配体的远端氢键供体的性质， 使蛋白质失活的滴定基团的身份， 在α-内消旋位置的方向上配体的空间倾斜，和 两个Tyr之间的远端氢键的性质可能是 在反应之前参与“封闭”基底腔。的影响 还将检查这些结构特征上的突变。的前景 还将探索细胞色素P450的确定性溶液核磁共振研究。是 我希望对解决方案结构的理解有很大的提高 目标酶及其功能的关系将由 这些研究。