Dimicoli J L, Renaud A, Lestienne P, Bieth J G
J Biol Chem. 1979 Jun 25;254(12):5208-18.
19F NMR investigations of the interactions between elastase and the reversible inhibitors CF3CO-Ala3, CF3CO-Lys-Ala2 and CF3CO-Ala4 show that these peptides have a single mode of binding to the enzyme. Furthermore the results indicate that the CF3CO-group experiences the same environment in all of the reversible complexes formed with these inhibitors. This agrees with the higher affinity of these peptides for the enzyme as compared to the corresponding acetylated inhibitors and confirms our earlier hypothesis of the existence of a specific binding site for the CF3CO-group on the enzyme. The interactions between elastase and the irreversible inhibitors CF3CO-Alan chloromethyl ketone (n = 2, 3, 4, 5) and CF3CO-Lys-Ala4 chloromethyl ketone have been investigated by enzymatic measurements and 19F NMR spectroscopy. The kinetic constants k2 and KI describing the irreversible inhibition are significantly lower for all the CF3CO-peptide chloromethyl ketones with exception of CF3CO-Ala2 chloromethyl ketone, than for the corresponding acetylated ones. Moreover, 19F NMR spectroscopy enabled us to demonstrate, for the tri- and tetrapeptide derivatives, the parallel formation of reversible nonproductive enzyme.inhibitor complexes. The spectroscopic properties of these complexes are completely different from those of the irreversible complexes but are similar to those observed with the reversible complexes described above. In the case of the pentapeptide chloromethyl ketones, fast hydrolysis of the peptide or fast inactivation of the enzyme does not allow observations to be made but does not exclude the existence of reversible nonproductive complexes. In fact, their existence is strongly suggested by the enzyme reaction rate measurements. The similarity of the properties of all the reversible complexes, their striking differences with those of all the irreversible complexes, as well as their mutual exclusivity, permit the conclusion that the CF3CO-group does not bind at one of the classical S subsites of elastase.
对弹性蛋白酶与可逆抑制剂CF3CO - Ala3、CF3CO - Lys - Ala2和CF3CO - Ala4之间相互作用的19F核磁共振研究表明，这些肽与该酶具有单一结合模式。此外，结果表明，CF3CO基团在与这些抑制剂形成的所有可逆复合物中所处环境相同。这与这些肽相对于相应乙酰化抑制剂对该酶具有更高亲和力相一致，并证实了我们之前关于该酶上存在CF3CO基团特异性结合位点的假设。通过酶促测量和19F核磁共振光谱研究了弹性蛋白酶与不可逆抑制剂CF3CO - 丙氨酰氯甲基酮（n = 2、3、4、5）和CF3CO - Lys - Ala4氯甲基酮之间的相互作用。除CF3CO - Ala2氯甲基酮外，所有CF3CO - 肽氯甲基酮的描述不可逆抑制作用的动力学常数k2和KI均显著低于相应的乙酰化酮。此外，19F核磁共振光谱使我们能够证明，对于三肽和四肽衍生物，可逆非生产性酶 - 抑制剂复合物会同时形成。这些复合物的光谱性质与不可逆复合物的完全不同，但与上述可逆复合物所观察到的相似。对于五肽氯甲基酮，肽的快速水解或酶的快速失活使得无法进行观察，但并不排除可逆非生产性复合物的存在。事实上，酶反应速率测量强烈表明了它们的存在。所有可逆复合物性质的相似性、它们与所有不可逆复合物性质的显著差异以及它们的相互排斥性，使得可以得出结论：CF3CO基团并非结合在弹性蛋白酶的经典S亚位点之一。
