Structure-Function Relationships of Pepstatin-insensitive Carboxyl Proteinases from Prokaryotes

原核生物胃酶抑素不敏感羧基蛋白酶的结构-功能关系

基本信息

批准号：
06660105
负责人：
ODA Kohei
金额：
$ 1.34万
依托单位：
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：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

It is well known that carboxyl proteinases are commonly inhibited by pepstatin^<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. On the contrary, we have isolated novel carboxyl proteinases from fungi, bacteria and also thermophilic bacteria based on their insensitivities to pepstatin, DAN and EPNP.These enzymes were tentatively named pepstatin- insensitve carboxyl proteinases.In this study, we aimed to identify the catalytic residues of pepstatin-insensitive carboxyl proteinases from prokaryote cells. We foucussed our studies on carboxyl proteinases from Pseudomonas sp.101 (PCP) and Xanthomonas sp.T-22 (XCP). PCP and XCP are the first and second carboxyl proteinases isolated from prokaryote cells. The primary structures of PCP and XCP does not have any homologous sturucture to those of aspartic … More proteinases (pepstatin-insensitive carboxyl proteinase) reported so far. Moreover, the well-conserved structure, -Asp**-Thr-Gly- (Asp** : catalytic residue) in the active center of aspartic proteinases was not observed.The following results were obtained.1.Identification of Catalytic Residues by Using Site-directed Mutagenesis TechniquePCP (372 amino acid residues) and XCP (398 amino acid residues) have 52% homology to each other. Based on the high sequence homology, eight amino acid residues for catalytic residues (aspartic or gultamic residues) were piked up, and all of them were mutated to alanine residues. We analyzed these alanine mutants for both auto-catalytic processing ability and proteinase activity. Consequently, D170, E217, E222, and D328 (PCP numbering) are strongly suggested to be the candidates for the catalytic residues. Probably, a pair of them, which are closely related in tertiary structure constitutes the catalytic residues.2.Identification of Catalytic Residues by Using Tyrostatin DerivativesIn 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 for PCP and XCP) from kitasatosporia sp.No.55. Based on the chemical structure, we succeeded in synthesizing a compeptive inhibitor, available for probing the catalytic residues of PCP (N-benzyloxycarbonyl-L-phenylalanine-2,3-epoxypropyl ester).Accordingly, we are in a position to identify the catalytic residues at both of DNA and protein levels. We hope that we will be able to identify the catalytic residues of PCP and XCP in 1996.1)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，因此被称为羧基蛋白酶。天冬氨酸蛋白酶。这些酶在一级和三级结构上都高度同源，相反，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。在这项研究中，我们的目的是从原核细胞中鉴定胃酶抑素不敏感的羧基蛋白酶的催化残基。假单胞菌 sp.101 (PCP) 羧基蛋白酶的研究PCP 和 Xanthomonas sp.T-22 (XCP) 是从原核细胞中分离出来的第一种和第二种羧基蛋白酶。 PCP 和 XCP 的主要结构与天冬氨酸蛋白酶（胃酶抑素不敏感）没有任何同源结构。羧基蛋白酶）此外，迄今为止报道了保守的结构，-Asp**-Thr-Gly-（Asp**：催化）。天冬氨酸蛋白酶活性中心未观察到残基。得到以下结果。 1.利用定点突变技术鉴定催化残基PCP（372个氨基酸残基）和XCP（398个氨基酸残基）有52%同源性基于彼此的高序列同源性，将催化残基（天冬氨酸或谷氨酸残基）的八个氨基酸残基挑出。我们分析了这些强丙氨酸突变体的自催化加工能力和蛋白酶活性。建议将 D170、E217、E222 和 D328（PCP 编号）作为候选。很可能，它们中的一对在三级结构上密切相关的残基构成了催化残基。2.鉴定使用酪抑素衍生物的催化残留物在我们尝试在活性中心研究中使用抑制剂时，我们从kitasatosporia sp.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，PCP和XCP的Ki=2.5 nM）。 No.55.根据化学结构，我们成功合成了一种竞争性抑制剂，可用于探测催化作用。 PCP（N-苄氧基羰基-L-苯丙氨酸-2,3-环氧丙酯）残基。因此，我们能够在DNA和蛋白质水平上鉴定催化残基。 1996.1）胃酶抑素，胃蛋白酶抑制剂2）中PCP和XCP的催化残基； 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。