PROLYL ENDOPEPTIDASE FROM PYROCOCCUS FURIOSUS

描述(摘自申请者的摘要)：这项拟议的研究 阐述了脯氨基的催化机制和生理作用。 超嗜热性白僵菌的内肽酶(PEPase)。脯氨酸基 内肽酶存在于细菌等不同的系统发育类群中， 植物、真菌和哺乳动物，但它们的功能尚不清楚。一直以来 假设Pro内肽酶在调节细胞周期中起作用 哺乳动物神经肽的半衰期，如催产素、加压素和 血管紧张素II，因为它们裂解和灭活这些多肽。这个 古生菌P.Furiosus中一种脯氨酰内肽酶的鉴定 最适生长温度为100℃，表明这些酶 在生活在截然不同的栖息地的有机体中发挥作用。 该基因已克隆到pET-11d和pET-11d中。 在大肠杆菌中过表达。重组蛋白的可用性 极大地方便了PEPase的特性，因为该酶很容易 从大肠杆菌中高产率纯化，该系统避免了困难 与纯化所需的大量呋喃假单胞菌有关 大量的PEPase。此外，这种酶具有很高的 耐高温，常温下耐降解。所宣布的目标 研究的内容包括通过测量来表征底物特异性 Kcat/Km值与一系列底物，研究催化作用 PEPase的机制，特别是关于存在的 丝氨酸蛋白酶特有的电荷传递系统，如 α-糜蛋白酶，评估切割生物活性哺乳动物的能力 多肽，从P.Furiosus细胞提取物中识别内源底物， 让本科生参与一次具有挑战性的研究经历。

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.