Structure-Function Relationships of Pepstation-insensitive Carboxyl Proteinase from Bacteria

细菌胃蛋白酶不敏感的羧基蛋白酶的结构-功能关系

基本信息

批准号：
04660125
负责人：
ODA Kohei
金额：
$ 1.34万
依托单位：
Faculty of Textile Science. Kyoto Institute of Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

项目摘要

It is well known that carboxyl proteinases are commonly inhibited by pepstain^<1)>, DAN^<2)>, and EPNP^<3)>, and their catalytic residues are composed of two aspartic acid residues. Thus, carboxyl proteinases are termed aspartic proteinases. These enzymes are highly homologous in both the primary and tertiary structures.We have isolated novel carboxyl proteinases from frugi, bacteria and also thermophilic bacteria based on their insensitivities to pepstatin, DAN and EPNP.These enzymes were tentatively named pepstatin-insensitve carboxyl proteinases. In one of our studies, the primary structure of carboxyl proteinase B (consisting of 204 amino acids) from a fungus Scytalidum lignicolum has been established, and one of the catalytic residues of the enzyme was clarified to be Glu-53. This is the first report on glutamic proteinase. It seemed probable that the pepstain-insensitive carboxyl proteinases are not aspartic proteinases but glutamic proteinases. To confirm this possibility, we fo … More cussed our studies on a pepstatin-insensitive carboxyl proteinase from Pseudomonas sp. No. 101(PCP), which is the first carboxyl proteinase isolated from prokaryote cells. The primary structure of PCP has been determined to be a single polypeptide composed of 372 amino acid residues with one disulfide bridge. PCP does not have any homologous structure to those of aspartic proteinases reported so far. Moreover, the well-conserved structure, -Asp^<**>-Thr-Gly-(Asp^<**> : catalytic residue) in the active center of aspartic proteinases was not observed.In this study, the following results wera obtained.1. Identification of Catalytic Residues In our attempt to use inhibitor in the study of active center, we had isolated a novel inhibitor. tyrostatin (N-isovaleryl-tyrosyl-leucyl-tyrosinal, Ki = 2.5Nm) from Kitasatosporia sp.No.55. Based on the cmemical structure. we succeeded in synthesizing a compeptive inhibitor, available for probing the catalytic residues of PCP(N-benzyloxycarbonyl-L-phenyl-atanine-2,3-epoxypropyl ester).2. Analysis of PCP Gene We determined the whole DNA sequence of the PCP gene(about 3 Kbp). It was elucidated that PCP is composed of prepro part protein (215 amino acid residues) and mature protein (372 amino acid residues). Primary structure of the mature protein was identical to that chemically determined previouly. It was suggested that the propart protein plays important roles in the activation as well as secretion through the double layr of the cell.Accordingly, it is ready now to study the structure-function relationships, especially the catalytic residues on both side of protein and DNA level.1) pepstatin, pepsin inhibitor ; 2) DAN, diazoacetyl-DL-norleucine methylester ; 3) EPNP, 1,2-epoxy-3-(p-nitrophenoxy) propane. Less

众所周知，羧基蛋白酶通常被胃蛋白酶^<1)>、DAN^<2)>和EPNP^<3)>抑制，并且它们的催化残基由两个天冬氨酸残基组成。这些酶在一级和三级结构上都高度同源。我们从真菌、细菌和植物中分离出了新型羧基蛋白酶。嗜热细菌基于其对胃酶抑素、DAN 和 EPNP 不敏感。这些酶暂时被命名为胃酶抑素不敏感羧基蛋白酶。在我们的一项研究中，来自真菌 Scytalidum lignicolum 的羧基蛋白酶 B（由 204 个氨基酸组成）的一级结构具有以下特征。已建立，并且该酶的催化残基之一被澄清为Glu-53。这是关于谷氨酸蛋白酶的第一份报告，看来胃酶抑素不敏感的羧基蛋白酶不是天冬氨酸蛋白酶，而是谷氨酸蛋白酶。为了证实这种可能性，我们对来自假单胞菌的胃酶抑素不敏感的羧基蛋白酶进行了研究。 No. 101(PCP)，是第一个从原核细胞中分离出来的羧基蛋白酶的一级结构。 PCP已被确定为由372个氨基酸残基组成的具有一个二硫键的单一多肽，与迄今为止报道的天冬氨酸蛋白酶不具有任何同源结构，而且，-Asp^<**结构非常保守。天冬氨酸蛋白酶活性中心未观察到>-Thr-Gly-(Asp^<**>：催化残基)。本研究得到以下结果： 1．催化残留物的鉴定在我们尝试在活性中心研究中使用抑制剂时，我们从Kitasatosporia sp.No.55中分离出一种新型抑制剂（N-异戊酰-酪氨酰-亮氨酰-酪氨酸，Ki = 2.5Nm）。我们成功地合成了一种竞争性抑制剂，可用于探测催化残基。 PCP(N-苄氧基羰基-L-苯基-丙氨酸-2,3-环氧丙酯)。2.PCP基因分析我们确定了PCP基因的全DNA序列(约3Kbp)，阐明了PCP由以下物质组成。前原部分蛋白质（215个氨基酸残基）和成熟蛋白质（372个氨基酸残基）的一级结构与之前化学测定的相同。有人认为propart蛋白在细胞双层的激活和分泌中发挥着重要作用。因此，现在可以研究其结构与功能关系，特别是蛋白质和DNA水平两侧的催化残基.1) 胃酶抑素，胃蛋白酶抑制剂；2) DAN，重氮乙酰基-DL-正亮氨酸甲酯；3) EPNP， 1,2-环氧-3-(对硝基苯氧基)丙烷较少。