Quantitative characterization of furin specificity - Energetics of substrate discrimination using an internally consistent set of hexapeptidyl methylcoumarinamides
Quantitative characterization of furin specificity - Energetics of substrate discrimination using an internally consistent set of hexapeptidyl methylcoumarinamides
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DOI：
10.1074/jbc.274.33.23229
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发表时间：
1999-08-13
影响因子：
4.8
通讯作者：
Fuller, RS
中科院分区：
文献类型：
--
作者：
Krysan, DJ;Rockwell, NC;Fuller, RS
呋喃是枯草杆菌相关真核加工酶中kexin/Furin家族的一种必需的哺乳动物前蛋白加工酶，与底物的成熟有关，涉及发育、信号、凝血和致病。我们用一系列多肽甲基香豆酰胺底物研究了Furin专一性的能量学，与以往的报道相比，我们发现Furin可以裂解这些底物，其动力学与扩展肽和生理底物相当，其中最好的是六肽甲基香豆胺，Furin显示k(CAT)/K-m值大于10(6)M-1 S(-1)Furin对六肽底物有显著的抑制作用，但不是四肽底物。对多肽类呋喃类药物评价意义的观察。对P-1、P-2和P-4上的Furin和Kex2识别的定量比较表明，虽然P-1上的相互作用对这两种酶的催化作用有相似的贡献，但Furin对P-2识别的依赖程度降低了10倍，而对P-4识别的依赖程度却增加了10-100倍。呋喃最近被证明表现出P-6识别，我们发现这种相互作用对催化的贡献类似于1.4千卡/摩尔，与P-4残基的性质无关。我们还表明，P-2和P-6的有利残基将补偿P-1或P-4的次优残基，对Furin和Kex2的定量分析明显区分了枯草杆菌蛋白相关酶的加工和降解成员识别底物的性质。
Furin, an essential mammalian proprotein processing enzyme of the kexin/furin family of subtilisin-related eukaryotic processing proteases, is implicated in maturation of substrates involved in development, signaling, coagulation, and pathogenesis, We examined the energetics of furin specificity using a series of peptidyl methylcoumarinamide substrates, In contrast to previous reports, we found that furin can cleave such substrates with kinetics comparable to those observed with extended peptides and physiological substrates, With the best of these hexapeptidyl methylcoumarinamides, furin displayed k(cat)/K-m values greater than 10(6) M-1 s(-1) Furin exhibited striking substrate inhibition with hexapeptide but not tetrapeptide substrates, an observation of significance to the evaluation of peptide-based furin inhibitors. Quantitative comparison of furin and Kex2 recognition at P-1, P-2, and P-4 demonstrates that whereas interactions at P-1 make comparable contributions to catalysis by the two enzymes, furin exhibited a similar to 10-fold lesser dependence on P-2 recognition but a 10-100-fold greater dependence on P-4 recognition. Furin has recently been shown to exhibit P-6 recognition and we found that this interaction contributes similar to 1.4 kcal/mol toward catalysis independent of the nature of the P-4 residue. We have also shown that favorable residues at P-2 and P-6 will compensate for less than optimal residues at either P-1 or P-4, The quantitative analysis of furin and Kex2 specificity sharply distinguish the nature of substrate recognition by the processing and degradative members of subtilisin-related proteases.