Tanizawa K, Kasaba Y, Kanaoka Y
J Biochem. 1980 Feb;87(2):417-27. doi: 10.1093/oxfordjournals.jbchem.a132762.
Trypsin [EC 3.4.21.4]-catalyzed hydrolysis of "inverse substrates" was investigated kinetically. "Inverse substrates" for trypsin are specific substrates in which the arrangement of the site-specific group is reversed compared to that of the normal substrate, e.g., a cationic center is included in the leaving group instead of being in the acyl moiety (Tanizawa, K., Kasaba, Y., & Kanaoka, Y. (1977) J. Am. Chem. Soc. 99, 4485-4488). Acyl enzyme intermediates formed specifically from these substrates are advantageous for the mechanistic analysis of trypsin action, since the cationic group liberated from the acyl moiety can no longer exhibit specific interaction with the enzyme binding site in the subsequent deacylation stage. Remarkable rate acceleration at the deacylation step was observed on adding amidinium or ammonium compounds. The effects of the size of the acyl moiety and the charged molecule on the acceleration were examined. Latent properties of 1-butylamine as an activator were found in the present study. Based on these observations, it is suggested that a cationic molecule which can be well accommodated together with the acyl group within the active center cleft causes rate enhancement, with associated conformational changes.
对胰蛋白酶[EC 3.4.21.4]催化的“反向底物”水解反应进行了动力学研究。胰蛋白酶的“反向底物”是特定的底物，其中位点特异性基团的排列与正常底物相比是相反的，例如，阳离子中心包含在离去基团中而不是在酰基部分（谷泽，K.，笠场，Y.， & 金冈，Y.（1977年）《美国化学会志》99，4485 - 4488）。由这些底物特异性形成的酰基酶中间体有利于对胰蛋白酶作用机制的分析，因为从酰基部分释放的阳离子基团在随后的脱酰基阶段不再能与酶结合位点表现出特异性相互作用。在加入脒鎓或铵化合物时，观察到脱酰基步骤有显著的速率加速。研究了酰基部分的大小和带电分子对加速作用的影响。本研究发现了1 - 丁胺作为激活剂的潜在特性。基于这些观察结果，表明能够与酰基一起很好地容纳在活性中心裂隙内的阳离子分子会导致速率增强，并伴有相关的构象变化。
