Changes in solvation during DNA binding and cleavage are critical to altered specificity of the EcoRI endonuclease

Restriction endonucleases such as EcoRI bind and cleave DNA with great specificity and represent a paradigm for protein–DNA interactions and molecular recognition. Using osmotic pressure to induce water release, we demonstrate the participation of bound waters in the sequence discrimination of substrate DNA by EcoRI. Changes in solvation can play a critical role in directing sequence-specific DNA binding by EcoRI and are also crucial in assisting site discrimination during catalysis. By measuring the volume change for complex formation, we show that at the cognate sequence (GAATTC) EcoRI binding releases about 70 fewer water molecules than binding at an alternate DNA sequence (TAATTC), which differs by a single base pair. EcoRI complexation with nonspecific DNA releases substantially less water than either of these specific complexes. In cognate substrates (GAATTC) k(cat) decreases as osmotic pressure is increased, indicating the binding of about 30 water molecules accompanies the cleavage reaction. For the alternate substrate (TAATTC), release of about 40 water molecules accompanies the reaction, indicated by a dramatic acceleration of the rate when osmotic pressure is raised. These large differences in solvation effects demonstrate that water molecules can be key players in the molecular recognition process during both association and catalytic phases of the EcoRI reaction, acting to change the specificity of the enzyme. For both the protein–DNA complex and the transition state, there may be substantial conformational differences between cognate and alternate sites, accompanied by significant alterations in hydration and solvent accessibility.

诸如EcoRI的限制性内切酶（restriction endonucleases）可通过极高的特异性结合并切割DNA，是研究蛋白质-DNA相互作用与分子识别的经典范式。本研究通过渗透压（osmotic pressure）诱导水释放的方法，证实了结合水参与了EcoRI对底物DNA的序列识别过程。溶剂化状态的改变，在引导EcoRI进行序列特异性DNA结合的过程中发挥关键作用，同时也对催化阶段的位点识别至关重要。通过测定复合物形成过程中的体积变化，我们发现：在同源识别序列（GAATTC）处，EcoRI结合所释放的水分子比在仅存在单个碱基对差异的替代DNA序列（TAATTC）处少约70个。EcoRI与非特异性DNA结合所释放的水分子数量，远少于上述两种特异性结合复合物的情况。对于同源识别底物（GAATTC），随着渗透压升高，催化速率常数k(cat)会下降，这表明切割反应过程伴随约30个水分子的结合。而对于替代底物（TAATTC），反应过程则伴随约40个水分子的释放，这一点可通过渗透压升高时反应速率的显著加速得到证实。溶剂化效应的这些显著差异表明，水分子在EcoRI反应的结合与催化两个阶段的分子识别过程中均发挥关键作用，进而改变该酶的底物特异性。无论是蛋白质-DNA复合物还是反应过渡态，同源位点与替代位点之间均可能存在显著的构象差异，同时伴随水合状态与溶剂可及性的显著改变。