一些重要的位点特异性内切核酸酶的反应动力学
Reaction kinetics of some important site-specific endonucleases.
作者信息
Hinsch B, Kula M R
出版信息
Nucleic Acids Res. 1981 Jul 10;9(13):3159-74. doi: 10.1093/nar/9.13.3159.
Reaction kinetics of the site-specific endonucleases BamHI, BgIII, C1aI, EcoRI, HpaII, PstI, SaII, SmaI, and XorII were investigated employing some frequently used substrates. Six of these enzymes could be analyzed under steady-state conditions. Kinetic data were obtained from progress curves applying an integrated Michaelis-Menten equation. KM ranged from 4 x 10(-9) M to 4 x 10(-11) M. Activities also spanned two orders of magnitude. In the case of C1aI the analysis of the pre-steady-state kinetics ("burst reaction") allowed the assessment of several rate constants. The rate-limiting step is the very slow dissociation of the enzyme-product complex (0.22 min(-1)). This complex is formed from the enzyme-bound nicked intermediate at a rate of 1.7 min(-1). The introduction of the first cut is again faster by a factor of about 6. SmaI and XorII resembled C1aI in their kinetics. The burst reaction can be used for the easy and unambiguous determination of molar concentrations of site-specific endonucleases in any preparation, which is free of non-specific DNases.
利用一些常用底物研究了位点特异性内切酶BamHI、BgIII、C1aI、EcoRI、HpaII、PstI、SaII、SmaI和XorII的反应动力学。其中六种酶可在稳态条件下进行分析。通过应用积分米氏方程从进程曲线获得动力学数据。米氏常数（KM）范围为4×10⁻⁹ M至4×10⁻¹¹ M。活性也跨越了两个数量级。对于C1aI，对稳态前动力学（“爆发反应”）的分析允许评估几个速率常数。限速步骤是酶 - 产物复合物非常缓慢的解离（0.22 min⁻¹）。该复合物由与酶结合的带切口中间体以1.7 min⁻¹的速率形成。第一次切割的引入再次快约6倍。SmaI和XorII在动力学上类似于C1aI。爆发反应可用于轻松且明确地测定任何不含非特异性DNase的制剂中位点特异性内切酶的摩尔浓度。
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