Electronic/Molecular Structure of Enzyme Heme Pockets

描述：(申请人提供)本提案涉及试验性 分子和分子的定量表征方法 中等大小(30-80 kDa)活性中心的电子结构 含有血红素的酶，以了解其机制(过氧化物酶与。 加氧酶)和立体特异性(阿尔法与贝塔、伽马或三角洲-中位血红素 裂解)与底物的反应。水溶液核磁共振波谱研究 顺磁状态下的血红素酶的活性部位非常适合于 提供此类活动站点的详细描述，其中利用了 光谱色散的增加显著地促进了 对活性中心残基的拆分，以及丰富的新型结构 超精细位移中包含的信息是造成这种位移的原因 分散。方法论方法将更加强调量化 30-40 kDa低自旋铁的结构研究(核间距离) 酶及其对大78的指配和结构分析的推广 Kda)低自旋，铁质，以及更强的顺磁性，30-40 kDa高自旋， 亚铁血红素酶。 预计方法学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 对大量其他顺磁性化合物的核磁共振研究具有重要意义 其他研究实验室目前正在研究的金属蛋白。 最优开发的核磁共振策略将被应用于详细确定 突变和结扎对血红素空洞性质的影响 44 kDa辣根过氧化物酶底物结合口袋的研究 由共价血红素形成的血红素电子结构与 78 kDa乳过氧化物酶中的蛋白质基质。结扎剂和结扎剂的研究 底物结合的人血红素加氧酶将专注于协调明显的 在溶液中连接的络合物与在溶液中的结构差异 晶体中未连接的复合体相对于远端的位置 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，确切的来源 配体在α-介孔位置方向的空间倾斜，以及 两个酪氨酸之间可能存在的远端氢键的性质 参与在反应前“关闭”衬底空腔。的影响 这些结构特征的突变也将被检查。的前景 还将探索细胞色素P450的最终溶液核磁共振研究。它是 预计对解决方案结构的了解会有相当大的提高 目标酶及其与其功能的关系将由 这些研究。

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.