Ogasawara W, Kobayashi G, Okada H, Morikawa Y
Department of Bioengineering, Nagaoka University of Technology, Niigata, Japan.
J Bacteriol. 1996 Nov;178(21):6288-95. doi: 10.1128/jb.178.21.6288-6295.1996.
Two kinds of dipeptidyl aminopeptidase I (DAP I [cathepsin C])-like activities which hydrolyze Gly-Phe-p-nitroanilide (Gly-Phe-pNA) were detected in Pseudomonas sp. strain WO24. They were purified and characterized. The isolated enzymes, named DAP BII and DAP BIII, were revealed to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing. DAP BII was estimated to have a molecular mass of 150,000 Da by gel filtration and a subunit size of 73,000 Da by SDS-PAGE, indicating it to be a homodimer. The molecular mass of DAP BIII was evaluated to be approximately 60,000 Da by gel filtration and 69,000 Da by SDS-PAGE, indicating that it is monomeric. The isoelectric points of DAP BII and DAP BIII were 6.1 and 5.0, and their optimal pHs were 8.0 and 8.5 to 9.0, respectively. The result of peptide mapping for DAP BII and DAP BIII showed that these enzymes consist of different components. Both enzymes were completely inhibited by diisopropylphosphofluoride but not by general thiol inhibitors, indicating that they are serine proteases. DAP BII and DAP BIII hydrolyzed Gly-Phe-pNA but not Gly-Arg-pNA, both of which are model substrates for mammalian DAP I. Despite these shared activities toward DAP I, DAP BII released dipeptides from Ala-Ala-pNA and Lys-Ala-4-methylcoumarinamide (a substrate for DAP II), whereas DAP BIII did not hydrolyze either of these compounds and was presumed to prefer substrates composed of bulky, hydrophobic amino acids at P1 and P1' positions. In addition, DAP BII showed no endopeptidase activity, whereas DAP BIII possessed the activity on N-terminally blocked peptide derivatives besides exopeptidase activity. Assays performed with bioactive peptides such as angiotensin I and neuromedin N as substrates indicate that DAP BII has a considerably broader substrate specificity than DAP BIII and is able to hydrolyze an X-Pro bond, an imido bond that few peptidases and no known DAPs can cleave. These characteristics, namely, substrate specificities, molecular mass, pI, peptide mapping, pH optimum, and effect of inhibitors, suggested that the two DAPs purified in this work are distinct enzymes and do not belong to any of the previously reported DAP classes.
在假单胞菌属菌株WO24中检测到了两种可水解甘氨酰 - 苯丙氨酸 - 对硝基苯胺（Gly - Phe - pNA）的二肽基氨基肽酶I（DAP I [组织蛋白酶C]）样活性。它们被纯化并进行了特性分析。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）和等电聚焦显示，分离得到的酶，命名为DAP BII和DAP BIII，是均一的。通过凝胶过滤估计DAP BII的分子量为150,000 Da，通过SDS - PAGE估计其亚基大小为73,000 Da，表明它是同二聚体。通过凝胶过滤评估DAP BIII的分子量约为60,000 Da，通过SDS - PAGE评估为69,000 Da，表明它是单体。DAP BII和DAP BIII的等电点分别为6.1和5.0，它们的最佳pH分别为8.0和8.5至9.0。DAP BII和DAP BIII的肽图谱结果表明这些酶由不同成分组成。两种酶都被二异丙基磷酰氟完全抑制，但不被一般的硫醇抑制剂抑制，表明它们是丝氨酸蛋白酶。DAP BII和DAP BIII可水解Gly - Phe - pNA，但不能水解Gly - Arg - pNA，这两种都是哺乳动物DAP I的模型底物。尽管它们对DAP I有这些共同活性，但DAP BII可从丙氨酰 - 丙氨酸 - pNA和赖氨酰 - 丙氨酸 - 4 - 甲基香豆素酰胺（DAP II的一种底物）释放二肽，而DAP BIII不能水解这两种化合物中的任何一种，推测它更喜欢在P1和P1'位置由大的、疏水氨基酸组成的底物。此外，DAP BII没有内肽酶活性，而DAP BIII除了外肽酶活性外，对N端封闭的肽衍生物具有活性。以生物活性肽如血管紧张素I和神经降压素N为底物进行的测定表明，DAP BII的底物特异性比DAP BIII宽得多，并且能够水解X - Pro键，这是一种很少有肽酶和已知DAP能够切割的亚氨基键。这些特性，即底物特异性、分子量、pI、肽图谱、最佳pH和抑制剂的作用，表明在这项工作中纯化的两种DAP是不同的酶，不属于任何先前报道的DAP类别。
