前蛋白转化酶(PCs)参与多种疾病的发病,使它们成为有希望的药物靶标。大多数PC检测都是用很少的标准底物进行的,不管切割效率的差异。来自对底物类似物抑制剂的研究,合成了11种新型底物,并用5种PCs进行了表征。H-Arg-Arg-Tle-Lys-Arg-AMC是基于其kcat/KM值的最有效裂解的弗林蛋白酶底物。由于其较高的kcat值,选择乙酰基-Arg-Arg-Tle-Arg-Arg-AMC用于进一步测量,以证明这种改进的底物的益处。与我们的标准条件相比,它的使用允许弗林蛋白酶浓度降低10倍,这使得能够在经典条件下确定先前描述的紧密结合抑制剂的Ki值。在这种情况下,抑制剂MI-1148首次观察到缓慢结合行为.除了弗林,使用另外四个PC来表征这些底物。最有效切割的PC1/3底物是乙酰基-Arg-Arg-Arg-Tle-Lys-Arg-AMC。PC2和PC7的最高kcat/KM值被发现用于该底物的N-末端未受保护的类似物。尽管其他底物具有较高的kcat值。对于底物乙酰基-Arg-Arg-Tle-Lys-Arg-AMC观察到PC5/6A的最高效率。总之,我们已经确定了弗林的新底物,PC1/3、PC2和PC7适用于改进的酶动力学测量。
Proprotein convertases (PCs) are involved in the pathogenesis of various diseases, making them promising drug targets. Most assays for PCs have been performed with few standard substrates, regardless of differences in cleavage efficiencies. Derived from studies on substrate-analogue inhibitors, 11 novel substrates were synthesized and characterized with five PCs. H-Arg-Arg-Tle-Lys-Arg-AMC is the most efficiently cleaved furin substrate based on its kcat/KM value. Due to its higher kcat value, acetyl-Arg-Arg-Tle-Arg-Arg-AMC was selected for further measurements to demonstrate the benefit of this improved substrate. Compared to our standard conditions, its use allowed a 10-fold reduction of the furin concentration, which enabled Ki value determinations of previously described tight-binding inhibitors under classical conditions. Under these circumstances, a slow-binding behavior was observed for the first time with inhibitor MI-1148. In addition to furin, four additional PCs were used to characterize these substrates. The most efficiently cleaved PC1/3 substrate was acetyl-Arg-Arg-Arg-Tle-Lys-Arg-AMC. The highest kcat/KM values for PC2 and PC7 were found for the N-terminally unprotected analogue of this substrate, although other substrates possess higher kcat values. The highest efficiency for PC5/6A was observed for the substrate acetyl-Arg-Arg-Tle-Lys-Arg-AMC. In summary, we have identified new substrates for furin, PC1/3, PC2, and PC7 suitable for improved enzyme-kinetic measurements.