SPECTROSCOPIC STUDIES OF NONHEME IRON ENZYMES

非血红素铁酶的光谱研究

• 批准号: 2444686
• 负责人: EDWARD I SOLOMON
• 金额: $ 24.59万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2444686
• 关键词: active sites; alpha ketoglutarate; atomic absorption spectrometry; biomagnetism measurement; bleomycin; circular dichroism; electron spin resonance spectroscopy; enzyme substrate complex; gene mutation; hydroxylation; intermolecular interaction; lipoxygenase; oxidoreductase inhibitor; oxygenases; phenylalanine; protein structure function; pteridines

Mononuclear non-heme iron active sites are present in a wide range of enzymes involved in a variety of important biological functions requiring dioxygen. These include the lipoxygenases (fatty acid hydroperoxidation), bleomycin (anticancer drug involved in DNA cleavage), intra- and extradiol dioxygenases (degradation of aromatic rings), tetrahydropterin dependent hydroxylases (phenylalanine metabolism), and the alpha-ketoglutarate-dependent enzymes (substrate hydroxylation and ring closure). Both the ferrous and ferric oxidation states are involved in catalysis for different enzymes in this class, and substrate and oxygen bound intermediates have been observed. Much less is known about the active sites in these enzymes relative to heme systems as the non-heme iron centers are less spectroscopically accessible. The general goals of this research program have been to develop new spectroscopic methods for the investigation of non-heme iron active sites and to apply these methods to the study of the above enzymes to obtain molecular level insight into the catalytic mechanisms and to understand the differences in the active site geometric and electronic structure which relate to differences in O2 and substrate reactivity. These studies should also contribute significantly toward elucidating the similarities and differences between non-heme and heme iron sites. Studies thus far have emphasized variable-temperature variable-field magnetic circular dichroism (VTVH MCD) combined with other excited state spectroscopic methods to probe the geometric and electronic structure of non-heme ferrous and ferric sites and to define the unusual electronic structure of the NO complex of non-heme ferrous sites and its relation to possible oxygen intermediates. The specific aims of this proposal are to: l) Complete the development of VTVH MCD as a powerful probe of non-heme ferrous active sites; 2) Develop Fe L- edge X-ray absorption spectroscopy as a new probe of non-heme iron active sites, particularly for ferric centers and oxygen intermediates; 3) Extend studies on lipoxygenases to correlate to the two conflicting crystal structures and to the mammalian enzymes with systematic mutations which influence reactivity, define active site intermediates, and probe the interaction of the active site with inhibitors: 4) Continue studies on bleomycin to understand each of the steps of the catalytic mechanism, determine the effect on the iron site due to the interaction with DNA, and define the relation of bleomycin to heme and other non-heme iron systems; 5) Correlate results on the extradiol dioxygenases with parallel data on the intradiol dioxygenases to determine differences in substrate-iron active site interactions which relate to differences in activation and could influence the position of ring cleavage; 6) Define the interaction of the ferrous site of phenylalanine hydroxylase with the pterin cofactor, probe the key steps of the catalytic mechanism, and determine how mutations which affect the enzyme's reactivity change the active site and its interactions with cofactor and substrate; 7) Understand the interaction of the ferrous site of clavaminate synthase with the alpha-ketoglutarate cofactor, define the interaction of this binary complex with O2 and analogs, and determine the interactions of this site with different substrates which lead to hydroxylation versus ring closure chemistry.

单核非血红素铁活性位点存在于广泛的范围 参与各种重要生物学功能的酶 需要二氧化物。这些包括脂氧酶（脂肪酸 氢过氧化），博来霉素（参与DNA的抗癌药物 裂解），内部和插二二加氧酶（芳族降解 戒指），四氢翅蛋白依赖性羟化酶（苯丙氨酸 代谢）和α-酮戊二酸依赖性酶（底物 羟基化和环闭合）。亚铁和铁氧化 国家参与此类不同酶的催化， 已经观察到底物和氧结合的中间体。很多 对于这些酶相对于血红素的活性位点知之甚少 系统作为非血红素中心的系统在光谱上较少 可访问。该研究计划的一般目标是 开发新的光谱法来研究非血红素铁 主动地点并将这些方法应用于上述研究 酶以获得分子水平的洞察力，以了解催化机制 并了解活动地点几何形状的差异和 与O2和底物差异有关的电子结构 反应性。这些研究也应为 阐明非血红素和血红素之间的相似性和差异 铁地点。迄今为止的研究强调了可变温度 可变场磁圆二色性（VTVH MCD）结合 其他激发态光谱法探测几何和 非血红素和铁位的电子结构，并定义 非血红素非复合物的不寻常的电子结构 位点及其与可能的氧中间体的关系。具体 该建议的目的是：l）完成VTVH MCD的发展 作为非血红素活性部位的强大探测； 2）发展f- 边缘X射线吸收光谱作为非血红素铁的新探针 活性位点，特别是用于铁中心和氧中间体； 3）扩展对脂氧酶的研究，以与两个冲突相关联 具有系统的晶体结构和哺乳动物酶 影响反应性的突变，定义活性位点中间体， 并探测活性位点与抑制剂的相互作用：4） 继续研究博来霉素，以了解 催化机制，确定由于 与DNA相互作用，并定义博来霉素与血红素的关系 其他非血红素铁系统； 5）将引流的结果相关联 二氧酶具有与基因内二氧酶的平行数据 确定底物铁的主动位点相互作用的差异 与激活的差异有关，可能会影响 环裂； 6）定义了亚铁部位的相互作用 苯丙氨酸羟化酶与翼蛋白辅因子，探测关键步骤 催化机制，并确定如何影响突变 酶的反应性改变了活动位点及其与 辅因子和底物； 7）了解亚铁的相互作用 与α-酮戊二酸辅助因子的链球菌合酶的位点， 定义该二进制复合物与O2和类似物的相互作用，以及 确定该站点与不同基板的相互作用 导致羟基化与环闭合化学。