Minetti Conceição A S A, Remeta David P, Breslauer Kenneth J
Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, 610 Taylor Road, Piscataway, NJ 08854, USA.
Proc Natl Acad Sci U S A. 2008 Jan 8;105(1):70-5. doi: 10.1073/pnas.0710363105. Epub 2008 Jan 2.
We report a continuous hyperchromicity assay (CHA) for monitoring and characterizing enzyme activities associated with DNA processing. We use this assay to determine kinetic and thermodynamic parameters for a repair enzyme that targets nucleic acid substrates containing a specific base lesion. This optically based kinetics assay exploits the free-energy differences between a lesion-containing DNA duplex substrate and the enzyme-catalyzed, lesion-excised product, which contains at least one hydrolyzed phosphodiester bond. We apply the assay to the bifunctional formamidopyrimidine glycosylase (Fpg) repair enzyme (E) that recognizes an 8-oxodG lesion within a 13-mer duplex substrate (S). Base excision/elimination yields a gapped duplex product (P) that dissociates to produce the diagnostic hyperchromicity signal. Analysis of the kinetic data at 25 degrees C yields a K(m) of 46.6 nM for the E.S interaction, and a k(cat) of 1.65 min(-1) for conversion of the ES complex into P. The temperature dependence reveals a free energy (DeltaG(b)) of -10.0 kcal.mol(-1) for the binding step (E + S <--> ES) that is enthalpy-driven (DeltaH(b) = -16.4 kcal.mol(-1)). The activation barrier (DeltaG) of 19.6 kcal.mol(-1) for the chemical step (ES <--> P) also is enthalpic in nature (DeltaH = 19.2 kcal.mol(-1)). Formation of the transition state complex from the reactants (E + S <--> ES), a pathway that reflects Fpg catalytic specificity (k(cat)/K(m)) toward excision of the 8-oxodG lesion, exhibits an overall activation free energy (DeltaG(T)) of 9.6 kcal.mol(-1). These parameters characterize the driving forces that dictate Fpg enzyme efficiency and specificity and elucidate the energy landscape for lesion recognition and repair.
我们报道了一种用于监测和表征与DNA加工相关的酶活性的连续增色法（CHA）。我们使用该方法来确定针对含有特定碱基损伤的核酸底物的修复酶的动力学和热力学参数。这种基于光学的动力学分析利用了含损伤的DNA双链底物与酶催化的、损伤切除产物之间的自由能差异，该产物含有至少一个水解的磷酸二酯键。我们将该分析应用于双功能甲酰胺嘧啶糖基化酶（Fpg）修复酶（E），它能识别13聚体双链底物（S）中的8-氧代鸟嘌呤损伤。碱基切除/消除产生一个缺口双链产物（P），其解离产生诊断性增色信号。在25℃下对动力学数据的分析得出E.S相互作用的K(m)为46.6 nM，ES复合物转化为P的k(cat)为1.65 min(-1)。温度依赖性揭示了结合步骤（E + S <--> ES）的自由能（ΔG(b)）为-10.0 kcal.mol(-1)，该步骤由焓驱动（ΔH(b) = -16.4 kcal.mol(-1)）。化学步骤（ES <--> P）的活化能垒（ΔG）为19.6 kcal.mol(-1)，本质上也是焓性的（ΔH = 19.2 kcal.mol(-1)）。从反应物形成过渡态复合物（E + S <--> ES），这一途径反映了Fpg对切除8-氧代鸟嘌呤损伤的催化特异性（k(cat)/K(m)），其总体活化自由能（ΔG(T)）为9.6 kcal.mol(-1)。这些参数表征了决定Fpg酶效率和特异性的驱动力，并阐明了损伤识别和修复的能量格局。
