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PROLYL ENDOPEPTIDASE FROM PYROCOCCUS FURIOSUS

来自愤怒火球菌的脯氨酰内肽酶

基本信息

批准号：
6081455
负责人：
VALERIE J HARWOOD
金额：
$ 4.32万
依托单位：
UNIVERSITY OF SOUTH FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-06-01 至 2001-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6081455
关键词：
Archaea bacterial proteins endopeptidases enzyme mechanism enzyme substrate recombinant proteins

项目摘要

DESCRIPTION (Adapted from applicant's abstract): This proposed research addresses the catalytic mechanism and physiological role of the prolyl endopeptidase (PEPase) of the hyperthermophilic Pyrocossus furiosus. Prolyl endopeptidases are found in such diverse phylogenetic groups as bacteria, plants, fungi, and mammals, yet their function is unknown. It has been hypothesized that prolyl endopeptidases have a role in regulating the half-life of mammalian neuropeptides such as oxytocin, vasopressin, and angiotensin II, since they cleave and inactivate these peptides. The identification of a prolyl endopeptidases in P. furiosus, an archaeon with an optimal growth temperature of 100 deg C, demonstrates that these enzymes function in organisms that inhabit vastly different habitats. The prpA gene from P. furiosus has been cloned into pET 11d and overexpressed in E. coli. The availability of the recombinant protein greatly facilitates characterization of PEPase, as the enzyme is easily purified in high yield from E. coli, and this system avoids the difficulties associated with the large culture volumes of P. furiosus necessary to purify significant quantities of PEPase. In addition, the enzyme is highly thermostable and resists degradation at room temperature. The stated goals of the research include characterizing substrate specificity by measuring Kcat/km values with a series of substrates, studying the catalytic mechanisms of PEPase, particularly with respect to the existence of charge-relay systems characteristic of serine proteases such as alpha-chymotrypsin, assessing the ability to cleave bioactive mammalian peptides, identifying endogenous substrate from P. furiosus cell extracts, and involving undergraduates in a challenging research experience.

描述（改编自申请人摘要）：本拟议研究说明脯氨酰的催化机制和生理作用极端嗜热的强烈焦尾藻的内肽酶（PEG 4）。氨酰内肽酶存在于诸如细菌等不同的系统发生群中，植物、真菌和哺乳动物，但它们的功能尚不清楚。已经假设脯氨酰内肽酶在调节哺乳动物神经肽如催产素、加压素和血管紧张素II，因为它们切割和切割这些肽。的鉴定P. furiosus中脯氨酰内肽酶最佳生长温度为100摄氏度，表明这些酶在栖息于不同生境的生物体中发挥作用。已将来自P. furiosus的prpA基因克隆到pET 11 d中，在E.杆菌重组蛋白的可用性极大地促进了PEG 4的表征，因为酶很容易从E.大肠杆菌，该系统避免了困难，与纯化所需的大培养体积的P. furiosus相关大量的PE。此外，该酶具有高度热稳定的并且在室温下抗降解。既定目标包括通过测量来表征底物特异性， Kcat/km值与一系列基板，研究催化的机制，特别是关于存在的丝氨酸蛋白酶的特征性电荷中继系统， α-糜蛋白酶，评估切割生物活性哺乳动物的能力肽，鉴定来自P. furiosus细胞提取物的内源性底物，并让本科生参与具有挑战性的研究体验。