PYRIDOXAL AS A PROBE OF MOLECULAR FUNCTION

拟议的调查扩展了正在进行的关于磷酸吡哆醛的研究 (PLP)依赖于已知三级结构的酶。过去的工作 支持期导致天冬氨酸的cdna克隆的产生 转氨酶(AAT)及其作为完整前体的表达 在大肠杆菌中发现线粒体AAT。这种前体(PmAAT)是大量生产的 是第一个以线粒体为靶向的纯化前体 以分离的、稳定的蛋白质形式存在的蛋白质。此外，氨基 成熟酶的末端区域已被确定为对 PLP绑定环境。最近的工作也有助于 发展各种光谱和其他物理技术，如 分析单个PLP区域及其与蛋白质的亲和性的工具 结合部位，反之亦然。这些工具，以及特定的反PLP 这种具有良好特性的酶的抗体和选定的抑制剂将 用于新的研究。建议进行的调查包括： 1.研究与改制有关的结构性改变 首次将线粒体蛋白前体(PmAAT)分离成成熟的PLP- 依赖酶(MAAT)。2.利用pmAAT的定点突变技术 评估一些选定的单个残基对PLP结合和 蛋白质稳定性，并将前体结构的变化联系在一起 通过引入这种二聚体中的改变而引起的溶液 辅酶要求蛋白质的转位速度或程度 线粒体。3.确定维持的最小蛋白质大小 对PLP的亲和力，单个酶功能的保留，以及可能的 输入到线粒体。这些发现的影响可能会 PLP领域之外的后果，因为它们涉及三个基本问题 生物学问题：(A)主要蛋白质的分子结构 序列类比(前体/成熟蛋白质)；(B)前序列的影响 肽对前体的整体蛋白质结构和性质的影响；和(C) 线粒体是如何识别、输入和处理前体的。

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.