Structure-Function Relationships of Pepstatin-insensitive Carboxyl Proteinases from Prokaryotes

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。相反，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。在这项研究中，我们旨在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。我们重点研究了假单胞菌sp.101 （PCP）和黄单胞菌sp.T-22 （XCP）的羧基蛋白酶。PCP和XCP是从原核细胞中分离到的第一个和第二个羧基蛋白酶。PCP和XCP的一级结构与天冬氨酸的结构没有任何同源性，目前报道的蛋白酶（胃抑素不敏感的羧基蛋白酶）较多。此外，在天冬氨酸蛋白酶活性中心未发现保守性较好的-Asp**- thr - gly - （Asp**：催化残基）结构。得到了以下结果：利用位点突变技术鉴定催化残基epcp（372个氨基酸残基）和XCP（398个氨基酸残基）同源性为52%。基于高序列同源性，提取出8个催化残基（天冬氨酸残基和谷氨酸残基），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催化处理能力和蛋白酶活性。因此，D170、E217、E222和D328 （PCP编号）被认为是催化残基的候选物。催化残基可能由三级结构密切相关的一对组成。利用酪氨酸抑制素衍生物鉴定催化残基为了将抑制剂用于活性中心的研究，我们从kitasatosporia sp. 55中分离出一种新的抑制剂酪氨酸抑制素（n -isovaleryl- tyroyl -leucyl-tyrosinal，对PCP和XCP 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 （n -苯氧羰基- l-苯丙氨酸-2,3-环氧丙基酯）的催化残基。因此，我们能够在DNA和蛋白质水平上识别催化残基。我们希望能在1996年鉴定出PCP和XCP的催化残基。2) DAN，重氮乙酰- dl -去甲亮氨酸甲基lester；3) EPNP,1,2-环氧-3-（对硝基苯氧基）丙烷。少

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