Brautigam C A, Steitz T A
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
J Mol Biol. 1998 Mar 27;277(2):363-77. doi: 10.1006/jmbi.1997.1586.
A two-metal-ion catalytic mechanism has previously been proposed for several phosphoryl-transfer enzymes. In order to extend the structural basis of this mechanism, crystal structures of three single-stranded DNA substrates bound to the 3'-5' exonucleolytic active site of the large fragment of DNA polymerase I from Escherichia coli have been elucidated. The first is a 2.1 A resolution structure of a Michaelis complex between the large fragment (or Klenow fragment, KF) and a single-stranded DNA substrate, stabilized by low pH and flash-freezing. The positions and identities of the catalytic metal ions, a Zn2+ at site A and a Mg2+ at site B, have been clearly established. The structural and kinetic consequences of sulfur substitutions in the scissile phosphate have been explored. A complex with the Rp isomer of phosphorothioate DNA, refined at 2.2 A resolution, shows Zn2+ bound to both metal sites and a mispositioning of the substrate and attacking nucleophile. The complex with the Sp phosphorothioate at 2. 3 A resolution reveals that metal ions do not bind in the active site, having been displaced by a bulky sulfur atom. Steady-state kinetic experiments show that catalyzed hydrolysis of the Rp isomer was reduced only about 15-fold, while no enzyme activity could be detected with the Sp phosphorothioate, consistent with the structural observations. Furthermore, Mn2+ could not rescue the activity of the exonuclease on the Sp phosphorothioate. Taken together, these studies confirm and extend the proposed two-metal-ion exonuclease mechanism and provide a structural context to explain the effects of sulfur substitutions on this and other phosphoryl-transfer enzymes. These experiments also suggest that the possibility of metal-ion exclusion be taken into account when interpreting the results of Mn2+ rescue experiments.
先前已提出几种磷酰基转移酶的双金属离子催化机制。为了扩展该机制的结构基础，已阐明了与大肠杆菌DNA聚合酶I大片段的3'-5'核酸外切酶活性位点结合的三种单链DNA底物的晶体结构。第一种是大片段（或克列诺片段，KF）与单链DNA底物之间的米氏复合物的2.1埃分辨率结构，通过低pH和快速冷冻得以稳定。催化金属离子的位置和身份已明确确定，A位点为Zn2+，B位点为Mg2+。已探究了在可裂解磷酸基团中进行硫取代的结构和动力学后果。一种与硫代磷酸酯DNA的Rp异构体形成的复合物，在2.2埃分辨率下精制，显示Zn2+与两个金属位点结合，且底物和进攻亲核试剂位置错误。与Sp硫代磷酸酯形成的复合物在2.3埃分辨率下显示，金属离子未结合在活性位点，已被一个庞大的硫原子取代。稳态动力学实验表明，Rp异构体的催化水解仅降低约15倍，而Sp硫代磷酸酯未检测到酶活性，这与结构观察结果一致。此外，Mn2+无法挽救核酸外切酶对Sp硫代磷酸酯的活性。综上所述，这些研究证实并扩展了所提出的双金属离子核酸外切酶机制，并提供了一个结构背景来解释硫取代对该酶及其他磷酰基转移酶的影响。这些实验还表明，在解释Mn2+挽救实验结果时应考虑金属离子排除的可能性。
