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

• 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
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06660105/
• 关键词: Prokaryote; Pepstatin; Carboxyl proteinase; Acid proteinase; Aspartic proteinase; Catalytic residue (s); Amino acid sequence; Structure-Function Relationship; アスパルティックプロテアーゼ

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抑素、丹和EPNP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高度同源。相反，我们根据真菌、细菌和嗜热菌对胃抑素、DAN和EPNP不敏感的特性，从真菌、细菌和嗜热菌中分离到了新的羧基蛋白水解酶，并将这些酶暂定为胃抑素不敏感的羧基蛋白水解酶。我们对假单胞菌101(PCP)和黄单胞菌T-22(XCP)的羧基蛋白酶进行了研究。PCP和XCP是从原核细胞中分离到的第一和第二羧基蛋白水解酶。PCP和XCP的一级结构与天冬氨酸…的一级结构没有任何同源结构到目前为止，更多的蛋白酶(胃抑素不敏感的羧基蛋白酶)被报道。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**-Thr-Gly-(Asp**：催化残基)。结果如下：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)具有52%的同源性。在序列高度同源性的基础上，筛选出8个催化残基(天冬氨酸残基或谷氨酸残基)氨基酸残基，并全部突变为丙氨酸残基。我们分析了这些丙氨酸突变体的自动催化处理能力和蛋白酶活性。因此，D170、E217、E222和D328(五氯联苯编号)被强烈推荐为催化残留物的候选者。2.酪抑素类化合物对催化残基的鉴定为了将抑制剂用于活性中心的研究，我们从木霉55号菌株中分离到一种新的酪抑素抑制剂(N-异戊基-酪氨酰-亮氨基-酪氨酸，对PCP和XCP的Ki=2.5 nM)。基于化学结构，我们成功地合成了一种竞争性的抑制剂，可用于探测五氯酚((N-benzyloxycarbonyl-L-phenylalanine-2，3-epoxypropyl)的催化残基，相应地，我们能够在DNA和蛋白质水平上鉴定催化残基。我们希望我们能够在1996.1)胃蛋白酶抑制剂胃抑素；2)重氮乙酰基-DL-去亮氨酸甲酯；3)EPNP，1，2-环氧基-3-(对-硝基苯氧基)丙烷中确定五氯酚和六氯酚的催化残基。较少

项目成果

Kohei ODA: "Cloning,Nucleotide Sequence,and Expression of an Isovaleryl Pepstatinigsensitive Cafboxy Proteinase Gene from Pseudomonas sp 101" The Jurnal of Biological Chemistry. 269. 26518-26524 (1994)

Kohei ODA：“来自假单胞菌 sp 101 的异戊酰胃酶抑敏感 Cafboxy 蛋白酶基因的克隆、核苷酸序列和表达”《生物化学杂志》。

Kohei ODA,et al.: "Cloning,Nucleotide Sequence,and Expression of an Isovaleryl Pepstatin-insensitive Carboxyl Proteinase Gene from Pseudomonas sp. 101" The Journal of Biological Chemistry. 269. 26518-26524 (1994)

Kohei ODA 等人：“假单胞菌 101 中异戊酰胃酶抑素不敏感的羧基蛋白酶基因的克隆、核苷酸序列和表达”《生物化学杂志》。

Kaeko HAYASHI: "The Primary Structure of Pepstatin-Insensitive Carboxyl Proteinase Producsl by Pseudomonas sp No. 101" The Jurnal of Biochemistry. 118. 738-744 (1995)

Kaeko HAYASHI：“假单胞菌第 101 号对胃酶抑素不敏感的羧基蛋白酶产品的一级结构”《生物化学杂志》。

Kohei ODA,et al.: "Aspartic Proteinases : Structure,Function,Biology,and Biomedical Implications" Plenum Press,New York, 14 (1995)

Kohei ODA 等人：“天冬氨酸蛋白酶：结构、功能、生物学和生物医学意义”Plenum Press，纽约，14 (1995)

Kaeko Hayashi et al.: "The primary Structure of Pepstatin-Insensitive Carboxyl Proteinase Produced by Pseudomonas sp.No.101." The Journal of Biochemistry. 118. 738-744 (1995)

Kaeko Hayashi 等人：“假单胞菌 101 号产生的胃酶抑素不敏感羧基蛋白酶的一级结构。”