Elliott B W, Cohen C
J Biol Chem. 1986 Aug 25;261(24):11259-65.
We report here a procedure which results in the purification of an extracellular protease (designated Ps-1) from Pseudomonas aeruginosa. This enzyme cleaves fibrinogen so that the modified molecules form microcrystals and large single crystals. Precise knowledge of the Ps-1 cleavage sites is essential for the interpretation of the structural information provided by these crystals (Weisel, J. W., Stauffacher, C. V., Bullitt, E., and Cohen, C. (1985) Science 230, 1388-1391). Ps-1 is a single-chain polypeptide of Mr 30,000 which appears to function as a monomer. The pH optimum is 8-9. The activity of the protease is not decreased by inhibitors of thiol, carboxyl, or metallo proteases; the abolishment of activity by N alpha-p-tosyl-L-lysine chloromethyl ketone and the partial inhibition obtained with serine-reactive inhibitors suggests that Ps-1 may be a serine protease with an unusual active-site conformation. Studies with synthetic peptide substrates show that Ps-1 exhibits one of the most restricted specificities known for an endoproteinase: only peptide, ester, and amide bonds containing the carbonyl group of lysine are hydrolyzed. The limited specificity of Ps-1 should make it useful for other applications requiring the selective cleavage of proteins, such as sequence analysis and the isolation of domains.
我们在此报告一种从铜绿假单胞菌中纯化细胞外蛋白酶（命名为Ps - 1）的方法。这种酶可切割纤维蛋白原，使修饰后的分子形成微晶和大的单晶。精确了解Ps - 1的切割位点对于解释这些晶体所提供的结构信息至关重要（韦塞尔，J. W.，施陶费克，C. V.，布利特，E.，和科恩，C.（1985年）《科学》230，1388 - 1391）。Ps - 1是一种分子量为30,000的单链多肽，似乎以单体形式发挥作用。最适pH为8 - 9。该蛋白酶的活性不受巯基、羧基或金属蛋白酶抑制剂的影响；Nα - 对甲苯磺酰 - L - 赖氨酸氯甲基酮使活性丧失，以及丝氨酸反应性抑制剂产生的部分抑制作用表明，Ps - 1可能是一种具有不寻常活性位点构象的丝氨酸蛋白酶。对合成肽底物的研究表明，Ps - 1表现出一种已知的内切蛋白酶中最具限制性的特异性：只有含有赖氨酸羰基的肽键、酯键和酰胺键会被水解。Ps - 1有限的特异性使其在其他需要选择性切割蛋白质的应用中很有用，例如序列分析和结构域的分离。
