Markgren P O, Lindgren M T, Gertow K, Karlsson R, Hämäläinen M, Danielson U H
Department of Biochemistry, Uppsala University, BMC, Uppsala, Sweden.
Anal Biochem. 2001 Apr 15;291(2):207-18. doi: 10.1006/abio.2001.5025.
The interaction between HIV-1 protease and inhibitors has been studied with optical biosensor technology. Optimized experimental procedures and mathematical analysis permitted determination of association and dissociation rate constants. A sensor surface with native enzyme was unstable and exhibited a drift that was influenced by the binding of inhibitor. This was hypothesized to be due to a specific mechanism involving autoproteolysis and/or dimer dissociation. The use of a mutant predicted to be less susceptible to autoproteolysis (Q7K) than wild-type enzyme resulted in a minor effect on surface stability, while a completely stable surface was obtained by treatment of the immobilized enzyme with N-ethyl-N'-(dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide; the most stable surface was achieved by chemically modifying the Q7K enzyme. The stabilized surface was enzymatically active and the interaction with inhibitors was similar to that for native enzyme. Several of the inhibitors had very high association rates, and estimation of kinetic constants was therefore performed with a binding equation accounting for limited mass transport. Of the clinical inhibitors studied, saquinavir had the highest affinity for the enzyme, a result of the lowest dissociation rate. Although the dissociation rate for ritonavir was sixfold faster, the affinity was only twofold lower than that for saquinavir since the association rate was threefold faster. Nelfinavir and indinavir exhibited lower affinities relative to the other inhibitors, a consequence of a slower association for nelfinavir and a relatively fast dissociation for indinavir. These results show that biosensor-based interaction studies can resolve affinity into association and dissociation rates, and that these are characteristic parameters for the interaction between enzymes and inhibitors.
利用光学生物传感器技术研究了HIV-1蛋白酶与抑制剂之间的相互作用。优化的实验程序和数学分析方法能够测定结合和解离速率常数。带有天然酶的传感器表面不稳定，且表现出受抑制剂结合影响的漂移现象。据推测，这是由于涉及自身催化水解和/或二聚体解离的特定机制所致。使用一种预计比野生型酶更不易发生自身催化水解的突变体（Q7K），对表面稳定性的影响较小，而通过用N-乙基-N'-(二甲基氨基丙基)-碳二亚胺和N-羟基琥珀酰亚胺处理固定化酶可获得完全稳定的表面；通过对Q7K酶进行化学修饰可实现最稳定的表面。稳定化的表面具有酶活性，与抑制剂的相互作用与天然酶相似。几种抑制剂具有非常高的结合速率，因此使用考虑了有限质量传输的结合方程来估算动力学常数。在所研究的临床抑制剂中，沙奎那韦对该酶的亲和力最高，这是其解离速率最低的结果。尽管利托那韦的解离速率快六倍，但其亲和力仅比沙奎那韦低两倍，因为其结合速率快三倍。与其他抑制剂相比，奈非那韦和茚地那韦表现出较低的亲和力，这是奈非那韦结合较慢和茚地那韦解离相对较快的结果。这些结果表明，基于生物传感器的相互作用研究能够将亲和力解析为结合和解离速率，并且这些是酶与抑制剂之间相互作用的特征参数。
