Catalysis of Thiol-Disulfide Exchange

描述（由申请人提供）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，蛋白质在加速折叠的酶和抑制聚集的伴侣蛋白的帮助下折叠成其天然结构。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使用。我们的长期目标是了解和模仿折叠助剂的生物化学和酶学。蛋白质二硫异构酶（PDI）是一种折叠催化剂和内质网（ER）的伴侣，对蛋白质二硫化物的正确形成和异构化至关重要。提出以下具体目标。具体目标PDI特异性及其多域结构。PDI的多结构域结构对于催化异构化是必不可少的，但是底物相互作用对PDI识别和催化的作用尚未被确定。来自底物肽阵列的数据表明，PDI相互作用集中在对异构化构成障碍的带正电荷的底物位点上，这些位点位于多个PDI结构域上。该模型将通过在定义明确的PDI底物中诱变赖氨酸和精氨酸残基以及将PDI结构域缺失突变体与底物序列的肽阵列结合来进行测试。具体目标2。酵母内质网的异构化和还原途径。目前的内质网二硫化物形成模型表明，PDI形成和破坏底物二硫化物，氧化和还原的PDI都需要适当的折叠。最近发现的异构酶缺陷PDI突变体仍然支持野生型酵母生长，将用于测试维持内质网氧化还原平衡的模型。实验还将测试补充PDI活性的补偿途径，包括内质网中非必需的PDI同源物的诱导和更一般的未折叠蛋白反应。具体目标3。异构化的机理。使PDI成为有效异构化催化剂的因素尚不清楚。为了确定PDI是否提供了一个涉及还原和再氧化的更快的异构化途径，PDI将与牛胰腺胰蛋白酶抑制剂（BPTI）的特定折叠中间体一起呈现，这些中间体在没有PDI的情况下进行缓慢的分子内异构化。PDI突变体和野生型PDI的单次翻转实验将检测PDI是否通过改变机制提供更快的异构化。

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.