CATALYSIS OF LEADER PEPTIDASE

前导肽酶的催化

• 批准号: 2186318
• 负责人: ROSS E DALBEY
• 金额: $ 8.76万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-01 至 1998-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2186318
• 关键词: acidity /alkalinity; active sites; chemical kinetics; circular dichroism; conformation; crosslink; enzyme activity; enzyme inhibitors; enzyme mechanism; enzyme structure; enzyme substrate; exopeptidase; fluorescence spectrometry; fluorescent dye /probe; ionic strengths; mutant; nucleic acid sequence; protein engineering; protein isoforms; protein signal sequence; site directed mutagenesis; temperature

Leader peptidase is an intracellular protease that plays an important role in protein export and catalyzes the removal of the amino-terminal leader sequences from exported proteins. The mechanism of action of leader peptidase is unknown and all available data suggests that it may belong to an unknown class of proteinase. Recently, our laboratory has provided evidence that suggest that leader peptidase is a member of an unusual class of serine protease that utilizes a lysine residue for catalysis. In this research proposal, we propose 1) to probe the catalytic mechanism of leader peptidase using kinetics, 2) to characterize an active delta2-75 leader peptidase fragment, 3) to test the serine/lysine catalytic model of leader peptidase, 4) to identify the substrate-binding residues within leader peptidase, 5) to map out the active site region, and 6) to prepare a fluorogenic pre-protein substrate for purposes of developing a rapid, continuous assay of leader peptidase activity. Oligonucleotide-directed mutagenesis, gene cloning, protein chemistry, kinetics, fluorescence, and CD spectroscopy techniques will be used to achieve these specific aims. The X-ray structure of the delta2-75 leader peptidase will be determined in a collaborative project. These studies will also help in the design of bacterial antibiotics that are targeted against leader peptidase, which is essential to bacterial growth.

铅肽酶是一种重要作用的细胞内蛋白酶 在蛋白质出口并催化氨基末端领导者的去除 来自导出蛋白的序列。领导者的行动机制 肽酶是未知的，所有可用的数据都表明它可能属于 未知类别的蛋白酶。最近，我们的实验室提供了 表明领导肽酶是不寻常的成员的证据 使用赖氨酸残基进行催化的丝氨酸蛋白酶。 在 这项研究建议，我们建议1）探测 使用动力学的领导者肽酶，2）表征活跃的delta2-75 领导者肽酶片段，3）测试丝氨酸/赖氨酸的催化模型 领导者肽酶，4）确定内部底物结合残基 领导者肽酶，5）绘制活性位点区域，6）准备 荧光前蛋白质底物，目的是开发快速的， 连续测定铅肽酶活性。 寡核苷酸定向 诱变，基因克隆，蛋白质化学，动力学，荧光和 CD光谱技术将用于实现这些特定目标。 将确定Delta2-75领导肽酶的X射线结构 在一个协作项目中。 这些研究还将有助于设计 针对铅肽酶的细菌抗生素，即 细菌生长必不可少。

项目成果

Blood pressure, but not cerebrospinal fluid fentanyl concentration, predicts duration of labor analgesia from spinal fentanyl.

血压而非脑脊液芬太尼浓度可预测脊髓芬太尼分娩镇痛的持续时间。

• DOI: 10.1097/aln.0b013e3181c38c0b
• 发表时间: 2010
• 期刊: Anesthesiology
• 影响因子: 8.8
• 作者: Nelson,KennethE;Houle,TimothyT;Eisenach,JamesC
• 通讯作者: Eisenach,JamesC

Use of site-directed chemical modification to study an essential lysine in Escherichia coli leader peptidase.

使用定点化学修饰研究大肠杆菌前导肽酶中的必需赖氨酸。

• DOI: 10.1074/jbc.272.15.9994
• 发表时间: 1997
• 期刊: The Journal of biological chemistry
• 作者: Paetzel,M;Strynadka,NC;Tschantz,WR;Casareno,R;Bullinger,PR;Dalbey,RE
• 通讯作者: Dalbey,RE