Control Mechanisms of the Nitric Oxide Synthases

一氧化氮合成酶的控制机制

• 批准号: 6544498
• 负责人: DENNIS J STUEHR
• 金额: $ 34.97万
• 依托单位: CLEVELAND CLINIC FOUNDATION
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6544498
• 关键词: Raman spectrometry; calmodulin; catalyst; chemical kinetics; chimeric proteins; electron transport; enzyme activity; enzyme substrate complex; flavin adenine dinucleotide; flavin mononucleotide; flavins; fluorescence spectrometry; heme; isozymes; nitric oxide; nitric oxide synthase; oxidation reduction reaction; protein engineering; protein structure function; stop flow technique; tissue /cell culture

DESCRIPTION (provided by applicant): Three NOS isoforms evolved to function in human health and disease. Despite structural similarities, each NOS has a different catalytic profile that may broaden their biologic function. In NOS a flavin-containing reductase domain transfers NADPH electrons to a heme in the oxygenase domain and this enables NO synthesis. Catalytic regulation is complex because NO binds to the heme before being released from NOS. We developed a kinetic model that incorporates these and other facets. Our kinetic model reveals that unique catalytic profiles of the three NOS are due to differences in the parameters: Ferric heme reduction (kr), ferric heme-NO dissociation (kd), and oxidation rate of the ferrous heme-NO complex (kox). Although NOS reductase domains control kr and oxygenase domains control kd and kox, the mechanisms and structural basis are unclear. We will perform biochemical, mutagenesis, rapid kinetic, and biophysical experiments test hypotheses regarding how and why kr and kox are broadly regulated in NOS. Aim I. Determine how structural features regulate electron transfer in NOS reductase (NOSr) domains. Our new crystal structure of the intact nNOSr guides these studies: Characterize eNOS and nNOS chimeras in which discreet reductase subdomains are swapped. Examine importance of specific FMN module surface contacts. Determine function of FAD-FMN linker domain. Test importance of H-bonding and other interactions of C-terminal extension in controlling flavin reduction. Compare flavin reduction potentials, electron distribution, and FMN shielding eNOSr and nNOSr and in their isolated FMN modules. Determine how FMN binding site structure controls thermodynamics and electron transfer.Aim II. Examine mechanism and structure function of ferrous heme-NO oxidation by 02 (kox). kox is key because it controls the speed of the futile cycle and also generates an uncharacterized N-oxide product that distinct from NO. Determine reaction mechanism, identify reaction intermediates and the immediate N-oxide product. Examine how NOS heme reduction potential influences kox and the reaction mechanism. Investigate role of N proximal heme loop in controlling kox.Aim III. Generate NOS with novel catalytic behaviors through protein engineering. Variant NOS create will possess combinations of kr and kox designed to greatly shift catalysis toward a futile cycle. These will test understanding of kinetic control mechanism and reveal how NOS products and function can change when key kinetic parameters deviate from their natural ranges.

描述（由申请人提供）：三种NOS同工型演变为在人类健康和疾病中发挥作用。尽管结构相似，但每个NOS都有不同的催化曲线，可以扩大其生物学功能。在NOS中，含黄素的还原酶结构域将NADPH电子转移到氧合酶结构域中的血红素中，这不能构成合成。催化调节很复杂，因为在从NOS释放之前没有与血红素结合。我们开发了一种结合这些方面和其他方面的动力学模型。我们的动力学模型表明，这三个NOS的独特催化谱归因于参数的差异：铁血红素还原（KR），铁血红素 - NO分离（KD）和氧化速率（KOX）。尽管NOS还原酶结构域控制KR和氧合酶结构域控制KD和KOX，但机理和结构基础尚不清楚。我们将进行生化，诱变，快速动力学和生物物理实验测试假设，以了解NOS中KR和KOX的方式以及为什么广泛调节。 AIM I.确定结构特征如何调节NOS还原酶（NOSR）域中的电子转移。我们完整的NNOSR的新晶体结构指导了这些研究：表征了eNOS和NNOS嵌合体，其中谨慎的还原酶亚域被交换了。检查特定FMN模块表面触点的重要性。确定FAD-FMN链接域的功能。测试H键的重要性和C末端扩展在控制黄素减少方面的重要性。比较黄素还原电位，电子分布以及FMN屏蔽ENOSR和NNOSR以及其分离的FMN模块。确定FMN结合位点结构如何控制热力学和电子传输。IAMII。通过02（KOX）检查了亚美蛋白氧化的机理和结构功能。 KOX是关键，因为它可以控制徒劳循环的速度，还会产生与NO不同的未表征的N氧化物产品。确定反应机制，鉴定反应中间体和直接的N-氧化物产物。检查NOS血红素还原电位如何影响KOX和反应机理。研究N近端血红素环在控制Kox.AIM III中的作用。通过蛋白质工程产生具有新型催化行为的NOS。变体NOS Create将拥有KR和KOX的组合，旨在将催化趋于徒劳的周期。这些将测试对动力学控制机制的理解，并揭示当关键动力学参数偏离其自然范围时，NOS产品和功能如何变化。