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

众所周知，羧基蛋白酶通常被胃蛋白酶抑制，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。这些酶在一级结构和三级结构上都有很高的同源性。我们根据Frugi、细菌和嗜热菌对胃抑素、DAN和EPNP不敏感的特性，从Frugi、细菌和嗜热细菌中分离到了新的羧基蛋白酶，这些酶暂时被命名为胃抑素不敏感的羧基蛋白酶。在我们的一项研究中，我们已经确定了木质青霉菌羧基蛋白酶B(由204个氨基酸组成)的一级结构，并明确了该酶的一个催化残基是Glu-53。这是有关谷氨酸蛋白酶的首次报道。推测不敏感的胃蛋白酶可能不是天冬氨酸蛋白酶，而是谷氨酸蛋白酶。为了确认这一可能性，我们向…进一步讨论了我们对来自假单胞菌的胃抑素不敏感的羧基蛋白酶的研究。第101号(PCP)，它是第一个从原核细胞中分离出来的羧基蛋白酶。PCP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，在天冬氨酸蛋白酶活性中心没有观察到保守的结构-Asp^&lt；**&gt；-Thr-Gly-(Asp^&lt；**&gt；)。催化残基的鉴定在我们尝试将抑制剂用于活性中心的研究中，我们分离出了一种新型的抑制剂。酪抑素(N-异戊基-酪氨酰-亮氨基-酪氨酸，Ki=2.5 nm)由北极子孢属55号菌株分离得到。基于化学结构。我们成功地合成了一种竞争性的缓蚀剂，可用于探测PCP(N-benzyloxycarbonyl-L-phenyl-atanine-2，3-epoxypropyl酯的催化残基)。PCP基因的分析我们测定了PCP基因的全长DNA序列(约3kbp)。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。该成熟蛋白的一级结构与先前化学测定的完全相同。因此，需要进一步研究其构效关系，特别是在蛋白质和DNA水平上的催化残基：1)胃蛋白酶抑制剂胃抑素；2)重氮乙酰去甲亮氨酸甲酯；3)EPNP，1，2-环氧基-3-(对硝基苯氧基)丙烷。较少

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