Catalysis of Thiol-Disulfide Exchange

硫醇-二硫化物交换的催化

• 批准号: 6992766
• 负责人: HIRAM F GILBERT
• 金额: $ 32.49万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 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 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6992766
• 关键词: SDS polyacrylamide gel electrophoresis; active sites; biomimetics; catalyst; chemical kinetics; endoplasmic reticulum; enzyme activity; enzyme substrate; high performance liquid chromatography; high throughput technology; molecular chaperones; oxidation reduction reaction; polymerase chain reaction; protein disulfide isomerase; protein folding; protein structure function; stereochemistry; thermodynamics; thiols; yeasts

DESCRIPTION (provided by applicant) Attaining the correct conformation and structure is essential for the proper function of proteins. In the cell, protein folding into its native structure is assisted by enzymes that accelerate folding and by chaperones that inhibit aggregation. Protein misfolding contributes to Alzheimer's disease, Parkinson's disease, prion-mediated infection, emphysema, and cystic fibrosis. Misfolding also limits the effective therapeutic use of recombinant proteins. Our long-term goal is to understand and mimic the biochemistry and enzymology of folding assistants. This proposal focuses on the in vitro and in vivo enzymology of protein disulfide isomerase (PDI), a folding catalyst and chaperone of the endoplasmic reticulum (ER) that is essential for the correct formation and isomerization of protein disulfides. The following specific aims are proposed. Specific Aim 1. PDI specificity and its multi-domain structure. The multidomain structure of PDI is essential for catalysis of isomerization, but substrate interactions that contribute to PDI recognition and catalysis have not been identified. Data from arrays of substrate peptides suggest a model in which PDI interactions are focused on positively charged substrate sites that pose barriers to isomerization and that these sites are located on multiple PDI domains. The model will be tested by mutagenesis of lysine and arginine residues in well-defined PDI substrates and the binding of domain deletion mutants of PDI to peptide arrays of substrate sequences. Specific Aim 2. Isomerization and reductive pathways in the yeast ER. The current model for ER disulfide formation suggests that PDI forms and breaks substrate disulfides and that oxidized and reduced PDI are both needed for proper folding. Recently discovered isomerase-deficient PDI mutants that still support wild-type yeast growth will be used to test models for maintaining ER redox balance. Experiments will also test for compensation pathways to supplement PDI activity, including the induction of non-essential PDI homologues in the ER and the more general unfolded protein response. Specific Aim 3. Mechanisms of isomerization. The factors that make PDI an effective catalyst of isomerization are not understood. To determine if PDI provides a faster pathway of isomerization involving reduction and reoxidation, PDI will be presented with specific folding intermediates of bovine pancreatic trypsin inhibitor (BPTI) which undergo slow, intramolecular isomerization in the absence of PDI. PDI mutants and single turnover experiments with wild-type PDI will detect if PDI provides faster isomerization by changing the mechanism. Mutations that alter the thiol/disulfide exchange kinetics and thermodynamics of the PDI active site will be used to determine which features of the chemistry of the active site contribute to catalysis.

说明（申请人提供）获得正确的构象和结构对于蛋白质的正常功能至关重要。在细胞中，蛋白质折叠成其天然结构是由加速折叠的酶和抑制聚集的分子伴侣辅助的。蛋白质错误折叠导致阿尔茨海默病、帕金森病、朊病毒介导的感染、肺气肿和囊性纤维化。错误折叠也限制了重组蛋白的有效治疗用途。我们的长期目标是了解和模拟折叠助剂的生物化学和酶学。该建议侧重于蛋白质二硫键异构酶（PDI）的体外和体内酶学，PDI是内质网（ER）的折叠催化剂和伴侣蛋白，对蛋白质二硫键的正确形成和异构化至关重要。提出了以下具体目标。具体目标1。PDI特异性及其多结构域结构。PDI的多结构域结构对于异构化的催化是必不可少的，但是还没有确定有助于PDI识别和催化的底物相互作用。来自底物肽阵列的数据表明了一种模型，其中PDI相互作用集中在带正电荷的底物位点上，这些位点对异构化构成障碍，并且这些位点位于多个PDI结构域上。将通过明确定义的PDI底物中赖氨酸和精氨酸残基的诱变以及PDI的结构域缺失突变体与底物序列的肽阵列的结合来测试该模型。具体目标2。酵母内质网中的异构化和还原途径。ER二硫化物形成的当前模型表明PDI形成并破坏底物二硫化物，并且氧化和还原的PDI都是正确折叠所需的。最近发现的异构酶缺陷的PDI突变体，仍然支持野生型酵母生长将被用来测试模型，以维持ER氧化还原平衡。实验还将测试补偿途径以补充PDI活性，包括在ER中诱导非必需的PDI同源物和更一般的未折叠蛋白质反应。具体目标3。异构化机制。使PDI成为异构化的有效催化剂的因素尚不清楚。为了确定PDI是否提供了涉及还原和再氧化的更快的异构化途径，PDI将与牛胰蛋白酶抑制剂（BPTI）的特异性折叠中间体一起呈现，其在不存在PDI的情况下经历缓慢的分子内异构化。PDI突变体和野生型PDI的单周转实验将检测PDI是否通过改变机制提供更快的异构化。 改变PDI活性位点的硫醇/二硫键交换动力学和热力学的突变将用于确定活性位点的化学特征中哪些有助于催化。