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将以： *了解结构如何影响金属酶活性位点的功能。 *确定结构，属性（例如自旋状态）和反应性之间是否存在相关性。我们将试图解决的问题包括： *需要哪些配体环境来稳定亚二氮水醇酶（NHASE）的低旋转状态？自旋状态对反应性有什么影响（如果有的话）？硫磺如何影响这些特性？胺在NHASE中扮演什么角色？ NHASE滋润亚硝酸盐的机制是什么？保守的ser-OH（或共同NHass中的thr-oH）在NHase机制中起什么作用？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）的超氧化物是否通过Fe（III）-OOH中间体进行？保守的谷氨酸在SOR机制中起什么作用（如果有的话）？抑制剂N3-和CN-如何抑制SOR活性？ CYS在SOR中的定位是否相对于开放结合位点，对促进功能很重要吗？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，并试图通过系统地改变配体的结构来将其与结构和特性（例如旋转状态和电子结构）相关联。 *在某些情况下，配体将掺入悬垂的酒精或鸟嘌呤臂，作为NHase附近丝氨酸和精氨酸的模型。 *将检查这些模型与亚trates 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根据动力学研究和热力学研究进行比较。 *反应性将在低温和EPR下通过分光光度法测量。 *动力学数据将使用停止流动技术和NMR线形分析获得。