Structure's Influence on Reactivity in Metalloenzymes

结构对金属酶反应活性的影响

• 批准号: 6991185
• 负责人: Julia A Kovacs
• 金额: $ 2.65万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-02-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6991185
• 关键词: active sites; alcohol dehydrogenase; chemical synthesis; cobalt; cyanides; cysteine; electron spin resonance spectroscopy; enzyme activity; enzyme complex; enzyme inhibitors; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate; hydro lyase; hydrogenase; iron; metalloenzyme; nuclear magnetic resonance spectroscopy; protein structure function; stop flow technique; synthetic enzyme

Research described in this proposal will be aimed at: * understanding how structure influences the function of metalloenzyme active sites. * determining if there is a correlation between structure, properties (such as spin-state), and reactivity. Questions we will be attempting to address include: * What ligand environment is needed to stabilize the low-spin state of nitrite hydratases (NHases)? What influence, if any, does spin-state have on reactivity? How do the sulfurs influence these properties? What role do the amidates play in NHase? What is the mechanism by which NHase hydrates nitrites? What role, if any, does the conserved Ser-OH (or Thr-OH in Co-NHases) play in the mechanism of NHase? What role, if any, do the conserved arginines play in the mechanism of NHase? Does superoxide reduction by superoxide reductases (SORs) proceed via an Fe(III)-OOH intermediate? What role, if any, does the conserved glutamate play in the mechanism of SOR? How do inhibitors N3- and CN- inhibit SOR activity? Is the positioning of the Cys in SOR, traps with respect to the open binding site, important in promoting function? We plan to explore these questions by: * examining the reactivity of synthetic model complexes, and attempting to correlate this with structure and properties, such as spin- state and electronic structure, by systematically altering the structure of our ligands. * ligands will, in some cases, incorporate pendent alcohol or guanidinium arms, as models for the nearby serine and arginines in NHase. * reactivity of these models with subtrates RCN (NHase), OH- (NHase), O2- (SOR) and inhibitors (N3- , NO, CN-, RC02-) will be examined. * reactivity of these models will be compared on the basis of kinetic and thermodynamic studies. * reactivity will be monitored spectrophotometrically, at low temperature, and by EPR. * kinetics data will be obtained using stopped-flow techniques, and NMR line-shape analysis.

该提案中描述的研究旨在：*了解结构如何影响金属酶活性位点的功能。 * 确定结构、性质（例如自旋态）和反应性之间是否存在相关性。我们将尝试解决的问题包括： * 需要什么配体环境来稳定亚硝酸盐水合酶 (NHase) 的低自旋状态？自旋态对反应性有什么影响（如果有的话）？硫如何影响这些特性？酰胺基在 NHase 中起什么作用？ NHase 水合亚硝酸盐的机制是什么？保守的 Ser-OH（或 Co-NHase 中的 Thr-OH）在 NHase 机制中发挥什么作用（如果有）？保守的精氨酸在 NHase 机制中起什么作用（如果有的话）？超氧化物还原酶 (SOR) 的超氧化物还原是否通过 Fe(III)-OOH 中间体进行？保守的谷氨酸在 SOR 机制中发挥什么作用（如果有的话）？抑制剂 N3- 和 CN- 如何抑制 SOR 活性？ SOR 中 Cys 的定位（相对于开放结合位点的陷阱）对于促进功能重要吗？我们计划通过以下方式探索这些问题： * 检查合成模型复合物的反应性，并尝试通过系统地改变配体的结构，将其与结构和性质（例如自旋态和电子结构）相关联。 * 在某些情况下，配体将包含悬垂的醇或胍臂，作为 NHase 中附近丝氨酸和精氨酸的模型。 * 将检查这些模型与底物 RCN (NHase)、OH- (NHase)、O2- (SOR) 和抑制剂（N3-、NO、CN-、RC02-）的反应性。 * 这些模型的反应性将在动力学和热力学研究的基础上进行比较。 * 反应性将在低温下通过分光光度法和 EPR 进行监测。 * 动力学数据将通过停流技术和 NMR 线形分析获得。