Tsu C A, Perona J J, Schellenberger V, Turck C W, Craik C S
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143.
J Biol Chem. 1994 Jul 29;269(30):19565-72.
Affinity-based purification and characterization of the collagenolytic serine protease 1 from Uca pugilator (fiddler crab) hepatopancreas shows that the enzyme cleaves the native bovine alpha 1(I) collagen chain carboxyl-terminal to Gln and Arg residues adjacent to the metallocollagenase site. Cleavage carboxyl-terminal to Leu residues is observed in the alpha 2(I) chain and at a secondary site in alpha 1(I). These sites correlate with the preferences observed toward p-nitroanilide substrates varying at the P1 position, for which the specificity (kcat/Km) is Arg > Leu, Phe, Lys > Gln > Ala. Furthermore, collagen cleavage after Gln was found exclusively between two Gln-Arg bonds. The P'1-P'3 specificity of collagenase, as determined by nucleophile acyl transfer, indicated a strong preference for Arg in the P'1 position. Crab collagenase cleaves peptide bonds adjacent to Leu and Gln at the P1 position more efficiently than trypsin, chymotrypsin, or elastase. Moreover, the efficiency of collagenase toward P1-Arg substrates is equivalent to that of trypsin. Crystals of crab collagenase have been grown complexed with the protein inhibitor ecotin. These crystals diffract to better than 2.8 A resolution and belong to the space group P3(2)21 with unit cell dimensions of a = b = 89.0 A, c = 291.7 A.
基于亲和作用从招潮蟹（提琴手蟹）肝胰腺中纯化和鉴定胶原olytic丝氨酸蛋白酶1，结果表明该酶在与金属胶原酶位点相邻的Gln和Arg残基的羧基末端切割天然牛α1(I)胶原链。在α2(I)链以及α1(I)的二级位点观察到在Leu残基羧基末端的切割。这些位点与在P1位置变化的对硝基苯胺底物所观察到的偏好相关，其特异性（kcat/Km）为Arg > Leu、Phe、Lys > Gln > Ala。此外，发现Gln之后的胶原切割仅发生在两个Gln-Arg键之间。通过亲核酰基转移测定的胶原酶的P'1-P'3特异性表明在P'1位置强烈偏好Arg。蟹胶原酶在P1位置比胰蛋白酶、胰凝乳蛋白酶或弹性蛋白酶更有效地切割与Leu和Gln相邻的肽键。此外，胶原酶对P1-Arg底物的效率与胰蛋白酶相当。蟹胶原酶的晶体已与蛋白质抑制剂依科汀复合生长。这些晶体的衍射分辨率优于2.8 Å，属于空间群P3(2)21，晶胞尺寸为a = b = 89.0 Å，c = 291.7 Å。
