Structure-Function Relationships of Pepstatin-insensitive Caroboxyl Protease produced by Pseudomonas sp. No. 101

假单胞菌产生的胃酶抑素不敏感的羧基蛋白酶的结构-功能关系。

基本信息

批准号：
02660124
负责人：
ODA Kohei
金额：
$ 1.28万
依托单位：
College of Agriculture, University of Osaka Prefecture
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

It is well known that carboxyl proteases are commonly inhibited by pepstatin^<1)>, DAN^<2)> and EPNP^<3)>, and their catalytic residues are composed of two aspartic acid residues. Thus, carboxyl proteases are termed aspartic proteases. These enzymes are highly homologous in both the primary and tertiary structures.We have isolated novel carboxyl proteases from fungi, bacteria and also thermophilic bacteria based on their insensitivities to pepstatin, DAN and EPNP. These enzymes were tentatively named pepstatin-insensitve carboxyl proteases. In one of our studies, the primary structure of carboxyl protease B(consisting of 204 amino acids)from a fungus Scytalidium lignicolum has been established, one of the catalytic residues of which was clarified to be Glu-53. This is the first report on glutamic protease. It seemed probable that the pepstatin-insensitive carboxyl proteases are not aspartic proteases but glutamic proteases. To confirm this possibility, we focussed our studies on a peps … More tatin-insensitive carboxyl protease from Pseudonionas sp. No. 101(PCP), which is the the first carboxyl protease-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 proteases reported so far. Moreover, the well-conserved structure, -Asp*-Thr-Gly- in the active center of aspartic proteases was not observed.In this study, the following results were 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 = 2.5 nM)from Kitasatosporia sp. No. 55. Based on the chemical structure, we succeeded in synthesizing a competitive inhibitor, available for probing the catalytic residues of PCP(Carbobenzoxy-Tyr-O-CH_2-Epoxide).2. Analysis of PCP Gene We determined the whole DNA sequence of the PCP gene(abaout 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 previously. It was suggested that the propart protein plays important roles in the activation as well as secretion through the double layer 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酶抑素^<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，因此，称为羧基蛋白酶。这些酶在一级和三级结构上都高度同源。我们从真菌、细菌中分离出了新型羧基蛋白酶。以及嗜热细菌，因为它们对胃酶抑素、DAN 和 EPNP 不敏感，这些酶暂时被命名为胃酶抑素不敏感的羧基蛋白酶。在我们的一项研究中，来自真菌 Scytalidium 的羧基蛋白酶 B（由 204 个氨基酸组成）的一级结构。已经建立了 ligicolum，其催化残留物之一被澄清为Glu-53。这是关于谷氨酸蛋白酶的第一份报告，胃酶抑素不敏感的羧基蛋白酶似乎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，我们将研究重点放在对胃蛋白酶不敏感的羧基蛋白酶上。来自假单胞菌 No. 101(PCP)，这是第一个羧基PCP 的一级结构已被确定为由 372 个氨基酸残基组成的具有一个二硫键的单一多肽，并且与迄今为止报道的天冬氨酸蛋白酶没有任何同源结构。天冬氨酸蛋白酶活性中心未观察到保守的结构-Asp*-Thr-Gly-。本研究得到以下结果： 1．催化残留物的鉴定在我们尝试使用抑制剂进行活性中心研究时，我们从Kitasatosporia sp. 55 中分离出一种新型抑制剂，酪抑素（N-异戊酰-酪氨酰-亮氨酰-酪氨酸，Ki = 2.5 nM）。在化学结构上，我们成功合成了一种竞争性抑制剂，可用于探测催化残基PCP(CarboBenzoxy-Tyr-O-CH_2-Epride)。2.PCP基因的分析我们测定了PCP基因的全DNA序列(约3kbp)，阐明PCP由前体部分蛋白(215个氨基酸)组成。成熟蛋白的一级结构与之前化学测定的结构相同，这表明原蛋白在激活中发挥着重要作用。因此，现在可以研究结构与功能的关系，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。