Metal ions bound at the active site of the junction-resolving enzyme T7 endonuclease I

T7 endonuclease I is a nuclease that is selective for the structure of the four-way DNA junction. The active site is similar to those of a number of restriction enzymes. We have solved the crystal structure of endonuclease I with a wild-type active site. Diffusion of manganese ions into the crystal revealed two peaks of electron density per active site, defining two metal ion-binding sites. Site 1 is fully occupied, and the manganese ion is coordinated by the carboxylate groups of Asp55 and Glu65, and the main chain carbonyl of Thr66. Site 2 is partially occupied, and the metal ion has a single protein ligand, the remaining carboxylate oxygen atom of Asp55. Isothermal titration calorimetry showed the sequential exothermic binding of two manganese ions in solution, with dissociation constants of 0.58 ± 0.019 and 14 ± 1.5 mM. These results are consistent with a two metal ion mechanism for the cleavage reaction, in which the hydrolytic water molecule is contained in the first coordination sphere of the site 1-bound metal ion.

T7核酸内切酶I（T7 endonuclease I）是一种对DNA四叉连接结构（four-way DNA junction）具有特异性识别能力的核酸酶，其活性位点与多种限制性内切酶（restriction enzymes）的活性位点结构相似。我们解析了带有野生型活性位点的T7核酸内切酶I的晶体结构。将锰离子扩散引入晶体后，每个活性位点处可观测到两处电子密度峰，由此确定了两个金属离子结合位点。位点1完全被占据，结合的锰离子由天冬氨酸55（Asp55）与谷氨酸65（Glu65）的羧基基团，以及苏氨酸66（Thr66）的主链羰基共同配位。位点2则为部分占据状态，该位点的金属离子仅拥有一个蛋白质配体，即天冬氨酸55的剩余羧基氧原子。等温滴定量热法（Isothermal titration calorimetry）实验表明，溶液中两个锰离子按顺序发生放热结合，其解离常数分别为0.58 ± 0.019 mM与14 ± 1.5 mM。上述结果与切割反应的双金属离子催化机制相符，该机制中水解水分子位于位点1结合的金属离子的第一配位层内。