Two types of novel dipeptidyl aminopeptidases from Pseudomonas sp strain WO24
Two types of novel dipeptidyl aminopeptidases from Pseudomonas sp strain WO24
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DOI：
10.1128/jb.178.21.6288-6295.1996
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发表时间：
1996-11-01
影响因子：
3.2
通讯作者：
Morikawa, Y
中科院分区：
文献类型：
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作者：
Ogasawara, W;Kobayashi, G;Morikawa, Y
两种二肽氨基肽酶Ⅰ水解Gly-Phe-对硝基苯胺的（DAP I [组织蛋白酶C]）样活性从假单胞菌WO 24菌株中检测到Gly-Phe-pNA，并对其进行了纯化和性质鉴定，经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳鉴定，所分离的酶为均一的DAP BII和DAP BIII通过SDS-PAGE和等电聚焦，通过凝胶过滤估计DAP BII的分子量为150，000 Da，通过SDS-PAGE估计DAP BII的亚基大小为73，000 Da，表明其为同源二聚体。经凝胶过滤和SDS-PAGE测定，DAP BIII的分子量约为60，000 Da，表明其为单体。DAP BII和DAP BIII的等电点分别为6.1和5.0，其最适pH分别为8.0和8.5 - 9.0，肽图分析结果表明，DAP BII和DAP BIII的酶由不同的组分组成，这两种酶都能被二异丙基磷氟化合物完全抑制，而不被一般的巯基抑制剂抑制，表明它们是丝氨酸蛋白酶，DAP BII和DAP BIII水解Gly-Phe-pNA，而不水解Gly-Arg pNA，这两种底物都是哺乳动物DAP I的模型底物。尽管DAP BII对DAP I具有相同的活性，但DAP BII从Ala-Ala-pNA和Lys-Ala-4-甲基香豆酰胺释放二肽（DAP II的底物），而DAP BIII不水解这些化合物中的任何一种，并且推测其优选由P1和P1'位置的大体积疏水性氨基酸组成的底物。此外，DAP BII没有表现出内肽酶活性，而DAP BIII除了具有外肽酶活性外，还具有对N-末端封闭的肽衍生物的活性。用生物活性肽如血管紧张素I和神经介肽N作为底物进行的测定表明，DAP BII具有比DAP BIII宽得多的底物特异性，并且能够水解X-Pro键，很少有肽酶和已知的DAP可以切割的亚氨基键，这些特征，即底物特异性、分子量、pi、肽图谱、最适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，不属于任何以前报道的DAP类别。
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 PI 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.