PYRIDOXAL AS A PROBE OF MOLECULAR FUNCTION

拟议的调查扩展了正在进行的磷酸吡哆醛研究 已知三级结构的PLP依赖性酶。 过去的工作 一段时间的支持导致生产的cDNA克隆的天冬氨酸 转氨酶（AAT）及其作为完整前体的表达 线粒体AAT在E.杆菌 这种前体（pmAAT）是大量生产的。 量，是第一个纯化的前体， 作为分离的稳定蛋白质可获得的蛋白质。 此外，氨基 成熟酶的末端区域已被确定为重要的 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， 用于分析单个PLP区域及其与蛋白质的接近度的工具 结合位点，反之亦然。 这些工具，沿着特定的抗PLP 抗体和选择的抑制剂，这种良好的表征酶，将 用于新的研究。 拟议的调查是： 1. 为了研究与转换有关的结构变化， 第一个分离的线粒体蛋白前体（pmAAT）转化为成熟的PLP- 依赖性酶（mAAT）。 2. 利用pmAAT的定点突变， 评估一些选定的单个残基对PLP结合的作用， 蛋白质的稳定性，并与前体结构的变化， 通过在这种二聚体和 辅酶需要蛋白的速率或程度，其易位到 线粒体 3. 为了确定最小蛋白质大小需要维持 对PLP的亲和力，保留单个酶功能，以及可能的 导入线粒体。 这些发现的影响可能会 超越PLP领域的后果，因为它们影响到三个基本的 生物学问题：（a）许多初级蛋白质的分子结构 序列类似物（前体/成熟蛋白）;（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;以及（c） 线粒体如何识别、输入和加工前体。

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.