BglⅠ限制性核酸内切酶与环状pBR322-DNA专一性和非专一性相互作用的动力学及热力学

 本文选择限制性核酸内切酶BglⅠ和pBR322-DNA为试验系统,用酶促反应的动力学和热力学方法来研究内切酶对环状DNA分子的专一性和非专一性结合及切割过程,求得了各限制位点的切割速度k及活化能E,各限制位点催化速度常数k_c,酶同限制位点专一性结合的平衡常数k_S非专一性结合的平衡常数k_N及其热力学参数△H,△S。研究表明:不论底物的构型如何(线状还是环状),内切酶都以相似的动力学和热力过程对其进行结合与切割。

Kinetics and Thermodynamics of Specific and Non-specific Interactions Between Bgll Restriction Endonuclease and pBR322-DNA

Cleavage of pBR322-DNA by BglI was studied at different temperatures and various incubation times. Kinetic constants and thermodynamic parameters of the reaction were evaluated.The similarity between linear and circular DNA in specific and non-specific interaction with the restriction endonuclease has been demonstrated by their kinetic constants and thermodynamic parameters. The specific binding is about 2 orders of magnitude stronger than non-specific binding. Ks is more dependent on temperature than KN. Complex formation is weakened with increasing temperature. The values of enthalpy and entropy show that both specific and non-specific binding need no activation energy and the "disorder" of the molecul is decreased with binding. The key element that limits the cleavage rates is KC.From the experienment results, it is evident that the recognition site adjacent to the A-T rich sequences has a low activation energy for cleavage and can be cleaved fast. This is probably due to the fact that the sequnnces adjacent to the recognition site may regulate thn activation threshold of the cleavage.