Shibuya Y, Yamamoto T, Morimoto T, Nishino N, Kambara T, Okabe H
Department of Laboratory Medicine, Kumamoto University Medical School, Japan.
Biochim Biophys Acta. 1991 Apr 29;1077(3):316-24. doi: 10.1016/0167-4838(91)90546-c.
Pseudomonas aeruginosa alkaline proteinase, which is a zinc-dependent bacterial endopeptidase, preferentially hydrolyzed Boc-Val-Leu-Lys-methylcoumarylamide (MCA) which was originally designed as a specific substrate of plasmin, a plasma serine proteinase. The hydrolytic capacity was resistant to tosyl-lysine chloromethylketone at a concentration as high as 1 mM, but was blocked by a treatment with metal chelator such as o-phenanthroline at the concentration of 5 mM. Kinetic parameters of the amidolytic reaction were Km = 21 microM, kcat = 0.067 s-1 and kcat/Km = 3190 M-1 s-1. A synthetic peptide inhibitor which bore a possible ligand for zinc atom at the carboxy terminal was designed. This inhibitor, Ac-Val-Leu-Lys-4-mercaptoanilide, blocked the amidolytic activity of the pseudomonal alkaline proteinase in a competitive manner with the dissociation constant (Ki) value of 24 microM. The results imply that P. aeruginosa alkaline proteinase must be an unusual zinc-dependent 'C (COOH)-type' endopeptidase, which hydrolyzes the peptide bond of certain amino acid residues at the carboxyl group side by specific recognition, like serine- and cysteine-proteinases. In comparison, P. aeruginosa elastase which is a typical 'N (NH2)-type' metalloproteinase did not hydrolyze all of the commercially available peptide-MCA substrates tested at the present study. P. aeruginosa alkaline proteinase also hydrolyzed natural substrates of plasmin, such as fibrin and fibrinogen, with similar specific activities to plasmin. The susceptible subunits of fibrinogen were the A-alpha and B-beta ones, in this order. P. aeruginosa alkaline proteinase also exhibited an anti-coagulant activity in human plasma attributed to the direct fibrinogenolytic function. Such potential anti-coagulant capacity of the P. aeruginosa alkaline proteinase might explain, at least partly, the most characteristic pathologic feature of the P. aeruginosa septicemia, hemorrhagic lesions with lacking thrombi (Fetzer, A.E. et al. (1967) Am. Rev. Respirat. Dis. 96, 1121-1130).
铜绿假单胞菌碱性蛋白酶是一种锌依赖性细菌内肽酶，它优先水解Boc - Val - Leu - Lys - 甲基香豆素酰胺（MCA），该物质最初被设计为血浆丝氨酸蛋白酶纤溶酶的特异性底物。其水解能力在高达1 mM的甲苯磺酰赖氨酸氯甲基酮浓度下具有抗性，但在5 mM的邻菲罗啉等金属螯合剂处理下会被阻断。酰胺水解反应的动力学参数为：Km = 21 μM，kcat = 0.067 s-1，kcat/Km = 3190 M-1 s-1。设计了一种在羧基末端带有可能的锌原子配体的合成肽抑制剂。这种抑制剂，Ac - Val - Leu - Lys - 4 - 巯基苯胺，以竞争性方式阻断铜绿假单胞菌碱性蛋白酶的酰胺水解活性，解离常数（Ki）值为24 μM。结果表明，铜绿假单胞菌碱性蛋白酶必定是一种不同寻常的锌依赖性“C（COOH）型”内肽酶，它通过特异性识别水解羧基侧某些氨基酸残基的肽键，类似于丝氨酸蛋白酶和半胱氨酸蛋白酶。相比之下，作为典型“N（NH2）型”金属蛋白酶的铜绿假单胞菌弹性蛋白酶在本研究中并未水解所有测试的市售肽 - MCA底物。铜绿假单胞菌碱性蛋白酶还能水解纤溶酶的天然底物，如纤维蛋白和纤维蛋白原，其比活性与纤溶酶相似。纤维蛋白原的敏感亚基依次为A - α和B - β亚基。铜绿假单胞菌碱性蛋白酶在人血浆中也表现出抗凝血活性，这归因于其直接的纤维蛋白原水解功能。铜绿假单胞菌碱性蛋白酶的这种潜在抗凝血能力可能至少部分解释了铜绿假单胞菌败血症最典型的病理特征，即缺乏血栓的出血性病变（费策尔，A.E.等人（1967年）《美国呼吸疾病评论》96，1121 - 1130）。
