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

众所周知，羧基蛋白酶通常被胃蛋白酶抑制剂^（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，羧基蛋白酶被称为天冬氨酸蛋白酶。这些酶在一级结构和三级结构上高度同源。我们从真菌、细菌和嗜热细菌中分离出了新的羧基蛋白酶，基于它们对胃蛋白酶抑制剂、DAN和EPNP不敏感。这些酶暂命名为胃蛋白酶抑制素不敏感的羧基蛋白酶。在我们的一项研究中，已经确定了来自真菌Scytalidium ligniculum的羧基蛋白酶B（由204个氨基酸组成）的一级结构，其中一个催化残基被澄清为Glu-53。这是关于谷氨酸蛋白酶的首次报道。胃蛋白酶抑制剂不敏感的羧基蛋白酶可能不是天冬氨酸蛋白酶，而是谷氨酸蛋白酶。为了证实这一可能性，我们将研究重点放在了一个peps上。 ...更多信息 Pseudonionas sp.No.101（PCP）是第一个从原核细胞中分离到的羧基蛋白酶。五氯苯酚的一级结构是由372个氨基酸残基和一个二硫键组成的单一多肽。PCP与已报道的天冬氨酸蛋白酶没有任何同源结构。此外，在天冬氨酸蛋白酶的活性中心没有观察到保守的结构-Asp*-Thr-Gly-.催化残基的鉴定在我们尝试在活性中心的研究中使用抑制剂时，我们已经从Kitasatosporia sp.No.55中分离出一种新的抑制剂，酪氨酸抑制素（N-异戊酰-酪氨酸酰-亮氨酰-酪氨酸醛，Ki = 2.5 nM）。根据PCP（Carbobenzoxy-Tyr-O-CH_2-Epoxide）的化学结构，成功合成了一种竞争性抑制剂，可用于PCP（Carbobenzoxy-Tyr-O-CH_2-Epoxide）催化残留的检测. PCP基因的分析测定了PCP基因的全序列（约3 kbp）。结果表明，PCP由215个氨基酸残基的前体蛋白和372个氨基酸残基的成熟蛋白组成。成熟蛋白的一级结构与先前化学测定的相同。因此，我们可以从蛋白质和DNA两个水平研究其结构与功能的关系：1）胃蛋白酶抑制剂pepstatin; 2）DAN; 3）EPNP。少

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