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

细菌胃蛋白酶不敏感的羧基蛋白酶的结构-功能关系

• 批准号: 04660125
• 负责人: ODA Kohei
• 金额: $ 1.34万
• 依托单位: Faculty of Textile Science. 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: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04660125/
• 关键词: Acid proteinase; Carboxyl proteinases; Aspartic proteinase; Pepstain-insensitive; S-PI-insensitive; Scytalidium type; Pepsin type; Structure-Function Relationship; 酸性プロテアーゼ

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

众所周知，羧基蛋白酶通常被pepstain^<1）>，dan^<2）>和epnp^<3）>抑制，并且它们的催化残留物由两个天冬氨酸残留物组成。这些酶在原始结构和三级结构中都是高度同源的。我们基于对pepstatin，dan和epnp的敏感性，分离出了来自frugi，细菌和嗜热细菌的新型羧基蛋白酶。这些酶被租用称为pepstatin不敏感的羧基蛋白酶。在我们的一项研究中，已经建立了来自真菌scytalidum木质糖的羧基蛋白酶B（由204个氨基酸组成）的主要结构，并阐明了酶的催化残留物之一，以glu-53。这是关于谷氨酸蛋白酶的第一个报告。脱蛋白不敏感的羧基蛋白酶可能不是天冬氨酸蛋白酶，而是谷氨酰胺蛋白酶。为了确认这种可能性，我们……更多地使我们对假单胞菌sp的pepstatin不敏感的羧基蛋白酶的研究更加困惑。第101号（PCP），这是从原核细胞中分离出的第一个羧基蛋白酶。 PCP的主要结构已被确定为由372个氨基酸残基组成的单个多肽，并具有一个二硫键。到目前为止，PCP对天冬氨酸蛋白酶的同源结构没有任何同源结构。 Moreover, the well-conserved structure, -Asp^<**>-Thr-Gly-(Asp^<**> : catalytic residence) in the active center of aspartic proteinases was not observed.In this study, the following results were obtained.1.在尝试使用抑制剂的活性中心研究中，鉴定催化残基，我们分离了一种新型的抑制剂。酪蛋白（N-异硫酸酪蛋白乳糖基 - 乳糖基，Ki = 2.5nm）来自Kitasatosporia sp.no.55。基于CMEMICAL结构。我们成功地合成了计算抑制剂，可用于探测PCP的催化残基（N-苯甲酰氧基碳苯甲苯l-l-苯基 - 苯胺-2,3--蛋白酶酯）.2。 PCP基因的分析确定了PCP基因的整个DNA序列（约3 kbp）。阐明了PCP由prepro部分蛋白（215个氨基酸保留）和成熟蛋白（372氨基酸保留）组成。成熟蛋白的一级结构与化学确定的预期相同。有人提出，Propart蛋白在细胞的双层中发挥重要作用，并在分泌中分泌。 2）DAN，重氮乙酰基-DL-甲氨甲基甲基酯； 3）EPNP，1,2-环氧-3-（p-硝基苯氧基）丙烷。较少的

项目成果

K.Oda, and S.Murao: Structure and Function of The Aspartic Proteinases Genetics, Structures, and Mechanisms ed. by B.M.Dunn "Pepstatin-insensitive Carboxyl Proteinases". Plenum Publishing Corporation, N.Y., 16 (1992)

K.Oda 和 S.Murao：天冬氨酸蛋白酶的结构和功能遗传学、结构和机制编辑。

K.Oda: "Structure and Function of The Aspartic Proteinases Genetics,Structures,and Mechanisms" ed.by B.M.Dunn Plenum Publishing Corporation,N.Y., 16 (1992)

K.Oda：“天冬氨酸蛋白酶遗传学、结构和机制的结构和功能”，B.M.Dunn Plenum Publishing Corporation 编辑，纽约，16 (1992)

K.Oda, T.Takahasni, Y.Tokuda, Y.Shibano and S.Takahashi: "Cloning, Sequencing, and Expression of Pepstatin-insensitve Carboxyl Proteinase Gene from Pseudomonas sp. No. 101" J.Biol.Chem.(under submission).

K.Oda、T.Takahasni、Y.Tokuda、Y.Shibano 和 S.Takahashi：“来自假单胞菌第 101 号的胃酶抑素不敏感羧基蛋白酶基因的克隆、测序和表达”J.Biol.Chem.（下）

K.Oda, H.Nakatani and B.M.Dunn: "Substrate Specificity, and Kinetic Properties of Pepstatin-insensitive Carboxyl proteinase from Pseudomonas sp. No.101" Biochim.Biophys.Acta. 1120. 208-214 (1992)

K.Oda、H.Nakatani 和 B.M.Dunn：“假单胞菌 No.101 中胃酶抑素不敏感的羧基蛋白酶的底物特异性和动力学特性”Biochim.Biophys.Acta。

K.Oda: "Substrate Specificity,and Kinetic Properties of Pepstatin-insensitive Carboxyl Proteinase from Pseudomonas sp.No.101" Biochim.Biophys.Acta. 1120. 208-214 (1992)

K.Oda：“来自假单胞菌属 sp.No.101 的胃酶抑素不敏感羧基蛋白酶的底物特异性和动力学特性”Biochim.Biophys.Acta。