Structure-Function of Novel Carboxyl Proteinases from Microorganisms

在这项研究中，我们的目的是从原核细胞中鉴定胃抑素不敏感的羧基蛋白酶的催化残基。本论文对假单胞菌的羧基蛋白酶进行了系统的研究。101(PCP)、黄单胞菌(Xanthomonas sp.)T-22(XCP)。凝结芽孢杆菌J-4(J-4)和新芽孢杆菌。MN-32(枯草杆菌溶血素)。它们的一级结构与迄今报道的天冬氨酸蛋白酶(胃抑素不敏感的羧基蛋白酶)没有任何相似之处。此外，保存完好的结构。-Asp*-Thr-Gly-(Asp*：催化残基)在天冬氨酸蛋白酶活性中心未观察到。取得了以下研究成果：1.用两套合成底物对枯草杆菌溶血素的底物专一性和亚基结构进行了研究。枯草杆菌溶血素的亚基结构与天冬氨酸蛋白酶的亚基结构不同。这些数据将发表在《生物化学》杂志上。用[^&lt；14&gt；C]…鉴定催化残渣更多的氧化亚锡用环氧化亚锡来鉴定催化残渣(S)。结果发现，环氧丁二烯分别与XCP的E75和D110残基结合。其中，PCP的E75残基(对应于PCP的E80残基)及其邻近区域保守，据此推测PCP的E80残基和XCP的E75残基分别作为底物结合位点参与了其催化作用。用二环己基二苯甲酰亚胺和特定的阻聚剂对S的催化残留物进行了化学修饰。Tyrostatin，发现[^&lt；14&gt；C]DCD分别与五氯苯酚的D140和E222残基结合。其中，E222残基(对应于XCP的E235残基)及其邻近残基在XCP中保守。此外，E222A突变体没有任何活性，而XE235A(对应于PCP的E222A)具有蛋白酶活性。基于这些数据。PCP的E222残基和XCP的E235残基可能参与了它们的催化作用。可能分别作为底物结合部位。凝结芽孢杆菌J-4羧基蛋白酶基因的克隆编号为歼-4。具有耐酒精和对胃抑素不敏感的特点。大部分基因已经被克隆和测序。在获得完整基因后，我们将尝试构建高效表达系统，并通过定点突变来确定催化残基。较少

In this study, we aimed to identify the catalytic residues of pepstatin-insensitive carboxyl proteinases from prokaryote cells. We focussed our studies on carboxyl proteinases from Pseudomonas sp. 101 (PCP), Xanthomonas sp. T-22 (XCP). Bacillus coagulans J-4 (J-4), and Bacillus novosp. MN-32 (kumamolysin). The primary structures of them does not have any similarities to those of aspartic 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. Substrate Specificity and Subsite Structure of KumamolysinSubstrate specificity of Kumamolysin was investigated by using two sets of synthetic substrates. The subsite structure of Kumamolysin was found to be different with those of aspartic proteinases. These data will be published in J.Biochem.2. Identification of Catalytic Residues by Using [^<14>C] … More Stylene OxideIn order to identify the catalytic residue(s) of XCP, [^<14>C] stylene oxide was used. It was found that the [^<14>C]stylene oxide was bound to E75 and D110 residues of XCP, respectively. Of them, E75 residue (corresponding to E80 residue of PCP) and its vicinities were conserved in PCP.Based on these data, E80 residue of PCP and E75 residue of XCP were thought to be involved in their catalytic function, as a substrate binding site, respectively.3. Identification of Catalytic Residues by Using [^<14>C] DCCDIn order to identify the catalytic residue(s) of PCP, chemical modification was carried out by using N.N'-dicyclohexylcafrbodiimide (DCCD) and specific inhibitor. tyrostatin, It was found that [^<14>C] DCCD was bound to D140 and E222 residues of PCP, respectively. Of them, E222 residue (corresponding to E235 residue of XCP) and its vicinities were found out to be conserved in XCP.Furthermore, E222A mutant had no any activity, whereas XE235A (corresponding to E222A for PCP) had proteinase activity. Based on these data. E222 residue of PCP and E235 of XCP were thought to be involved in their catalytic function. probably as a substrate binding site, respectively.4. Cloning of Carboxyl Proteinase J-4 Gene from Bacillus coagulansBacillus coagulans J-4 carboxyl proteinase. designated as J-4. is characterized as alcohol resistant and insensitive to pepstatin. Most of the gene has been cloned, sequenced. After getting the whole gene, we will try to construct a high expression system and determine the catalytic residues by site-directed mutagenesis. Less