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

众所周知，羧基蛋白酶通常被胃蛋白酶抑制剂^（1）>、DAN^（2）>和EPNP^（3）>抑制，并且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中高度同源。与此相反，我们从真菌、细菌和嗜热菌中分离到了一些对胃蛋白酶抑制剂、DAN和EPNP不敏感的新型羧基蛋白酶，暂命名为胃蛋白酶抑制剂不敏感羧基蛋白酶（pepstatin-insitve carboxylproteinases）。本文对假单胞菌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的第一和第二种羧基蛋白酶。PCP和XCP的一级结构与天冬氨酸没有任何同源性 ...更多信息 蛋白酶（胃蛋白酶抑制剂不敏感的羧基蛋白酶）。结果表明：1.利用定点突变技术对催化残基的鉴定PCP（372个氨基酸残基）和XCP（398个氨基酸残基）具有52%的同源性。基于序列高度同源性，挑取了8个催化残基（天冬氨酸或谷氨酸残基），并将它们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加工能力和蛋白酶活性。因此，强烈建议D170、E217、E222和D328（PCP编号）为催化残留物的候选物。我们从kitasatosporia sp.No.55中分离到一种新的酪氨酸抑制剂--酪氨酸抑制素（N-isovaleryl-tyrosyl-leucyl-tyrosinal，对PCP和XCP的Ki=2.5 nM）。根据PCP的化学结构，我们成功地合成了一种竞争性抑制剂，可用于PCP（N-苄氧羰基-L-苯丙氨酸-2，3-环氧丙酯）催化残基的检测，从而为在DNA和蛋白质水平上鉴定PCP的催化残基奠定了基础。1）胃蛋白酶抑制剂，胃蛋白酶抑制剂; 2）DAN，重氮乙酰-DL-正亮氨酸甲酯; 3）EPNP，1，2-环氧-3-（p-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