Abstract：
Human apurinic/apyrimidinic endonuclease APE1 catalyzes endonucleolytic hydrolysis of phosphodiester bonds on the 5\' side of structurally unrelated damaged nucleotides in DNA or native nucleotides in RNA. APE1 additionally possesses 3\'-5\'-exonuclease, 3\'-phosphodiesterase, and 3\'-phosphatase activities. According to structural data, endo- and exonucleolytic cleavage of DNA is executed in different complexes when the excised residue is everted from the duplex or placed within the intrahelical DNA cavity without nucleotide flipping. In this study, we investigated the functions of residues Arg177, Arg181, Tyr171 and His309 in the APE1 endo- and exonucleolytic reactions. The interaction between residues Arg177 and Met270, which was hypothesized recently to be a switch for endo- and exonucleolytic catalytic mode regulation, was verified by pre-steady-state kinetic analysis of the R177A APE1 mutant. The function of another DNA-binding-site residue, Arg181, was analyzed too; it changed its conformation when enzyme-substrate and enzyme-product complexes were compared. Mutation R181A significantly facilitated the product dissociation stage and only weakly affected DNA-binding affinity. Moreover, R181A reduced the catalytic rate constant severalfold due to a loss of contact with a phosphate group. Finally, the protonation/deprotonation state of residues Tyr171 and His309 in the catalytic reaction was verified by their substitution. Mutations Y171F and H309A inhibited the chemical step of the AP endonucleolytic reaction by several orders of magnitude with retention of capacity for (2R,3S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran-containing-DNA binding and without changes in the pH dependence profile of AP endonuclease activity, indicating that deprotonation of these residues is likely not important for the catalytic reaction.
摘要：
人无嘌呤/无嘧啶核酸内切酶APE1催化DNA中结构无关的受损核苷酸或RNA中天然核苷酸的5'侧磷酸二酯键的核酸内切水解。APE1还具有3'-5'-外切核酸酶，3'-磷酸二酯酶,和3'-磷酸酶活性。根据结构数据，当切除的残基从双链体外翻或置于螺旋内DNA腔内而不发生核苷酸翻转时，DNA的内切和核酸外切切割在不同的复合物中进行。在这项研究中,我们研究了残基Arg177，Arg181，Tyr171和His309在APE1核酸内切和核酸外切反应中的功能。残基Arg177和Met270之间的相互作用，最近被认为是内切和外切核酸催化模式调节的开关，通过R177AAPE1突变体的预稳态动力学分析验证。另一个DNA结合位点残基的功能，也分析了Arg181；当比较酶-底物和酶-产物复合物时，它改变了其构象。突变R181A显着促进了产物解离阶段，并且仅微弱地影响了DNA结合亲和力。此外，由于失去与磷酸基团的接触,R181A将催化速率常数降低了几倍。最后,残基Tyr171和His309在催化反应中的质子化/去质子化状态通过它们的取代来验证。Y171F和H309A突变将AP核酸内切反应的化学步骤抑制了几个数量级，并保留了（2R，3S）-2-（羟甲基）-3-羟基四氢呋喃-含DNA结合，并且AP内切核酸酶活性的pH依赖性曲线没有变化，这表明这些残留物的去质子化对催化反应可能不重要。
