Kim H J, Tamanoue Y, Jeohn G H, Iwamatsu A, Yokota A, Kim Y T, Takahashi T, Takahashi K
Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo.
J Biochem. 1997 Jan;121(1):82-8. doi: 10.1093/oxfordjournals.jbchem.a021575.
An extracellular metalloprotease was purified from the culture supernatant of Pseudomonas fluorescens strain KT1 to apparent homogeneity and shown to consist of a single polypeptide chain (M(r) 46,000-47,000). The enzyme was strongly inhibited by chelating agents such as EDTA and o-phenanthroline, and activated by certain detergents. Among the peptidyl 4-methylcoumaryl-7-amide (MCA) substrates examined, t-butyloxycarbonyl-Arg-Val-Arg-Arg-MCA was the best one. With this substrate, the enzyme exhibited a pH optimum of around pH 5.5 in the absence of Co2+ ions, whereas it showed two different pH optima (at pHs around 5.5 and 8-9) in the presence of Co2+ ions due to remarkable activation by Co2+ ions in the alkaline pH range. On the other hand, a single broad pH optimum of around 6 to 8 was obtained with some peptides in both the presence and absence of Co2+ ions, and no activation by Co2+ was observed. The enzyme showed trypsin-like specificity, preferentially cleaving certain arginyl peptide bonds, and hydrolyzed the basic protein, histone, most rapidly among various proteins examined. Partial amino acid sequence analysis revealed that the enzyme is highly homologous with proteases of the serralysin family, a group of zinc metalloproteases.
从荧光假单胞菌KT1菌株的培养上清液中纯化出一种细胞外金属蛋白酶，使其达到表观均一性，结果表明该酶由一条单一的多肽链组成（相对分子质量为46,000 - 47,000）。该酶受到螯合剂如EDTA和邻菲啰啉的强烈抑制，并被某些去污剂激活。在所检测的肽基4 - 甲基香豆素 - 7 - 酰胺（MCA）底物中，叔丁氧羰基 - 精氨酸 - 缬氨酸 - 精氨酸 - 精氨酸 - MCA是最佳底物。对于该底物，在没有Co2 +离子的情况下，该酶的最适pH约为5.5，而在有Co2 +离子存在时，由于Co2 +离子在碱性pH范围内具有显著的激活作用，它呈现出两个不同的最适pH（在pH约5.5和8 - 9处）。另一方面，无论有无Co2 +离子存在，使用某些肽时都可获得一个单一的较宽最适pH范围，约为6至8，且未观察到Co2 +的激活作用。该酶表现出胰蛋白酶样的特异性，优先切割某些精氨酰肽键，并且在检测的各种蛋白质中，对碱性蛋白质组蛋白的水解速度最快。部分氨基酸序列分析表明，该酶与解链酶家族的蛋白酶高度同源，解链酶家族是一组锌金属蛋白酶。
