Moore B M, Jalluri R K, Doughty M B
Department of Medicinal Chemistry, University of Kansas, Lawrence 66045, USA.
Biochemistry. 1996 Sep 10;35(36):11642-51. doi: 10.1021/bi952515m.
The nucleotide photoprobe 2-[(4-azidophenacyl)thio]-2'-deoxyadenosine 5'-triphosphate (1) was evaluated as a photoaffinity label of the DNA polymerase I Klenow fragment. Photolabel [3H]-1 covalently labeled the Klenow fragment with photolysis at 300 nm, reaching saturation at an approximate 1:1 mole ratio at 5.7 microM and with an EC50 (the effective concentration at 50% maximum photoincorporation) of about 0.74 microM. Saturating concentrations of poly(dA).(T)10 protect the Klenow fragment from [3H]-1 photoincorporation, and TTP at a concentration approximately equal to its KD for the free enzyme form shifts the dose-response curve for photoincorporation of [3H]-1 into the Klenow fragment by a factor of 2, indicating a competitive relationship between TTP and 1. Additionally, the photoincorporation of [3H]-1 into the Klenow fragment has an absolute requirement for magnesium, with no significant photoincorporation observed at concentrations of 1 up to 10 microM in the absence of magnesium. These results demonstrate that, as designed, photoprobe 1 binds to both the dNTP and a portion of the template-primer binding sites on the Klenow fragment. Photoaffinity labeling of the Klenow fragment by 1 yielded a single radiolabeled tryptic fragment which was isolated by HPLC; sequence analysis identified Asp732 in the peptide fragment Asp732-Ile733-His734-Arg735 as the site of covalent modification. Molecular modeling and complementary NMR analysis of the conformation of 1 indicated preferred C3'-exo and C2'-exo-C3'-endo symmetrical twist furanose ring puckers, with a high antibase conformation and a +sc C-5 torsional angle. Docking studies using Asp732 as an anchor point for the azide alpha-nitrogen on the photolabel indicate that the dNTP binding site is at the edge of the DNA binding cleft opposite the exonuclease site and that the template binding site includes helix O in the finger motif of the Klenow fragment.
核苷酸光探针2-[(4-叠氮苯甲酰基)硫代]-2'-脱氧腺苷5'-三磷酸（1）被评估为DNA聚合酶I Klenow片段的光亲和标记物。光标记物[3H]-1在300 nm光解条件下与Klenow片段共价结合，在5.7 microM时以约1:1的摩尔比达到饱和，其EC50（最大光掺入量的50%时的有效浓度）约为0.74 microM。饱和浓度的聚（dA）·（T）10可保护Klenow片段不被[3H]-1光掺入，当三磷酸胸腺嘧啶核苷（TTP）的浓度约等于其对游离酶形式的解离常数（KD）时，[3H]-1光掺入Klenow片段的剂量反应曲线会偏移2倍，这表明TTP与1之间存在竞争关系。此外，[3H]-1光掺入Klenow片段对镁有绝对需求，在无镁的情况下，当1的浓度高达10 microM时未观察到明显的光掺入。这些结果表明，如设计的那样，光探针1与Klenow片段上的dNTP以及模板-引物结合位点的一部分都有结合。1对Klenow片段进行光亲和标记产生了一个单一的放射性标记胰蛋白酶片段，通过高效液相色谱法（HPLC）分离得到；序列分析确定肽片段Asp732-Ile733-His734-Arg735中的Asp732为共价修饰位点。对1的构象进行分子建模和互补核磁共振分析表明，其呋喃糖环呈优选的C3'-外式和C2'-外式-C3'-内式对称扭曲构象，具有高反碱基构象和+sc C-5扭转角。以Asp732作为光标记物上叠氮基α-氮的锚定点进行对接研究表明，dNTP结合位点位于与核酸外切酶位点相对的DNA结合裂隙边缘，模板结合位点包括Klenow片段手指基序中的螺旋O。
