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

众所周知，羧基蛋白酶通常受到pepstatin^<1)>、DAN^<2)>和EPNP^<3)>的抑制，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。这些酶在一级和三级结构上都高度同源。我们已经从真菌、细菌和嗜热细菌中分离出了新的羧基蛋白酶，这些酶对胃抑素、DAN和EPNP不敏感。这些酶暂定名为胃抑素不敏感羧基蛋白酶。在我们的一项研究中，从真菌Scytalidium lignicolum中建立了羧基蛋白酶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。似乎胃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。为了证实这一可能性，我们将研究重点集中在从原核细胞中分离到的第一个羧基蛋白酶——Pseudonionas sp. No. 101（PCP）中分离到的一种对蛋白质不敏感的羧基蛋白酶上。PCP的一级结构是由372个氨基酸残基和一个二硫桥组成的单一多肽。PCP与目前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- asp *- thr - gly -结构。在本研究中，获得了以下结果：1。为了将抑制剂用于活性中心的研究，我们从Kitasatosporia sp. No. 55中分离到一种新的抑制剂tyrostatin（N-isovaleryl-tyrosyl-leucyl-tyrosinal, Ki = 2.5 nM）。基于化学结构，我们成功合成了一种竞争性抑制剂，可用于探测PCP（Carbobenzoxy-Tyr-O-CH_2-Epoxide）的催化残基。PCP基因分析我们确定了PCP基因的全DNA序列（约3kbp）。结果表明，PCP由前体蛋白（215个氨基酸残基）和成熟蛋白（372个氨基酸残基）组成。成熟蛋白的初级结构与之前化学测定的相同。提示该蛋白在细胞双层活化和分泌中起重要作用。因此，对其结构-功能关系的研究，特别是在蛋白质和DNA水平上的催化残基的研究已经做好了准备。1)胃抑素，胃蛋白酶抑制剂；2)DAN，重氮乙酰- dl -去甲亮氨酸甲基lester；3) EPNP 1.2 -epoxy-3 (P-nitrophenoxy)丙烷。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