Roychowdhury Anasuya, Szymanski Michal R, Jezewska Maria J, Bujalowski Wlodzimierz
Department of Biochemistry and Molecular Biology, The Sealy Center for Structural Biology and Molecular Biophysics, Sealy Center for Cancer Cell Biology, The University of Texas Medical Branch at Galveston, Texas 77555-1053, USA.
Biochemistry. 2009 Jul 28;48(29):6730-46. doi: 10.1021/bi9000529.
The kinetic mechanism of NTP binding and hydrolysis by the Escherichia coli replicative helicase, the DnaB protein, in the absence and presence of the single-stranded DNA (ssDNA), has been quantitatively examined using the rapid quench-flow technique, under single-turnover conditions. In the case of both the free helicase and the enzyme-ssDNA complexes, the mechanism is independent of the type of base of the cofactor or the DNA; the bimolecular association is followed by the reversible chemical hydrolysis and subsequent conformational transition of the enzyme-product complex. The NTP hydrolysis step is significantly faster for the purine than for the pyrimidine cofactor, both in the absence and in the presence of the DNA. The temperature effect indicates that the nature of intermediates of the purine nucleotide, ATP, is different from the nature of the analogous intermediates of the pyrimidine nucleotide, CTP. Nevertheless, both types of cofactors seem to approach a similar "exit" state at the end of the reaction. The effect of ssDNA on the kinetics of NTP hydrolysis depends on the type of nucleotide cofactor and the base composition of the DNA and is centered at the hydrolysis step. Homoadenosine ssDNA oligomers are particularly effective in increasing the hydrolysis rate. The allosteric signal from the DNA, which activates the NTP hydrolysis, comes predominantly from the strong DNA-binding subsite. The role of the weak DNA-binding subsite is to modulate the allosteric effect of the strong subsite. The significance of these results for the mechanism of the free energy transduction by the DnaB helicase is discussed.
利用快速淬灭流动技术，在单周转条件下，对大肠杆菌复制解旋酶DnaB蛋白在不存在和存在单链DNA（ssDNA）时NTP结合和水解的动力学机制进行了定量研究。对于游离解旋酶和酶-ssDNA复合物而言，该机制均与辅因子或DNA的碱基类型无关；双分子缔合之后是酶-产物复合物的可逆化学水解及随后的构象转变。无论是否存在DNA，嘌呤辅因子的NTP水解步骤都比嘧啶辅因子的显著更快。温度效应表明，嘌呤核苷酸ATP的中间体性质与嘧啶核苷酸CTP的类似中间体性质不同。然而，两种类型的辅因子在反应结束时似乎都接近相似的“出口”状态。ssDNA对NTP水解动力学的影响取决于核苷酸辅因子的类型和DNA的碱基组成，且集中在水解步骤。同型腺苷ssDNA寡聚物在提高水解速率方面特别有效。激活NTP水解的来自DNA的变构信号主要来自强DNA结合亚位点。弱DNA结合亚位点的作用是调节强亚位点的变构效应。讨论了这些结果对DnaB解旋酶自由能转导机制的意义。
