PYRIDOXAL AS A PROBE OF MOLECULAR FUNCTION

吡哆醛作为分子功能的探针

• 批准号: 3294320
• 负责人: MARINO MARTINEZ-CARRION
• 金额: $ 12.86万
• 依托单位: UNIVERSITY OF MISSOURI KANSAS CITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-09-01 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3294320
• 关键词: Escherichia coli; X ray crystallography; affinity chromatography; antibody formation; aspartate carbamoyltransferase; aspartate transaminase; cofactor; complementary DNA; conformation; electron probe spectrometry; enzyme inhibitors; enzyme mechanism; enzyme structure; enzyme substrate complex; genetic regulatory element; glutamate decarboxylase; ligands; membrane structure; mitochondrial membrane; molecular cloning; nutrition related tag; oligopeptides; protein biosynthesis; protein sequence; protein signal sequence; protein structure function; protein transport; pyridoxal phosphate; site directed mutagenesis; vitamin biosynthesis

The proposed investigation extends ongoing studies on pyridoxal phosphate (PLP)-dependent enzymes of known tertiary structure. Work during the past period of support led to the production of cDNA clones for aspartate transaminase (AAT) and their expression as intact precursor for mitochondrial AAT in E. coli. This precursor (pmAAT) was produced in large quantities and is the first purified precursor of a mitochondrial-targeted protein available as an isolated, stable protein. Furthermore, the amino terminal region of the mature enzyme has been identified as important for the PLP binding environment. Recent work also contributed to the development of a variety of spectroscopic and other physical techniques as tools for analyzing individual PLP regions and their proximity to protein binding sites and vice versa. These tools, along with specific anti-PLP antibodies and selected inhibitors of this well-characterized enzyme, will be used for the new studies. The proposed investigations are: 1. To study the structural changes associated with the conversion of the first isolated mitochondrial protein precursor (pmAAT) into a mature PLP- dependent enzyme (mAAT). 2. To use site-specific mutagenesis of pmAAT to assess the roles of some selected individual residues on PLP binding and protein stability, and to relate the changes in precursor structure in solution as induced by the introduction of alterations in this dimeric and coenzyme requiring protein to the rate or extent of its translocation into mitochondria. 3. To determine the minimum protein size needed to maintain affinity for PLP, retention of individual enzymatic functions, and possible import into mitochondria. The impact of these findings can have consequences beyond the PLP field, as they impinge into three fundamental biological problems: (a) Molecular structure for proteins of much primary sequence analogy (precursor/mature protein); (b) Effects of the presequence peptide on overall protein structure and properties of precursors; and (c) How precursors are recognized, imported and processed by mitochondria.

拟议的研究扩展了对吡啶还毒素的持续研究 （PLP）依赖性酶的已知三级结构的酶。 过去工作 支撑时期导致生产天冬氨酸的cDNA克隆 转氨酶（AAT）及其作为完整前体的表达 大肠杆菌中的线粒体AAT。 该前体（PMAAT）是在大的 数量，是线粒体靶向的第一个纯化前体 蛋白质可作为分离的稳定蛋白质可用。 此外，氨基 成熟酶的末端区域已被确定为重要 PLP结合环境。 最近的工作也有助于 开发各种光谱和其他物理技术 分析单个PLP区域及其与蛋白质接近的工具 绑定位点，反之亦然。 这些工具，以及特定的反PLP 该特征化酶的抗体和选定的抑制剂将 用于新研究。 拟议的调查是： 1。研究与转化相关的结构变化 首次分离的线粒体蛋白前体（PMAAT）成熟的PLP- 依赖酶（MAAT）。 2。使用PMAAT的位点特异性诱变 评估某些选定的单个残基在PLP结合中的作用和 蛋白质稳定性，并关联前体结构的变化 通过引入该二聚体和的变化引起的解决方案 辅酶需要蛋白质达到其易位的速率或程度 线粒体。 3。确定维持所需的最小蛋白质大小 对PLP的亲和力，保持单个酶功能的保留以及可能 进口到线粒体。 这些发现的影响可能会有 在PLP领域之外的后果，因为它们侵略了三个基本 生物学问题：（a）许多原发性蛋白质的分子结构 序列类比（前体/成熟蛋白）； （b）presequence的影响 肽的整体蛋白质结构和前体的特性； （c） 线粒体如何识别，进口和处理前体。