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首页> 外文期刊>Biochemistry >Identification of an Inhibitor Binding Site of Poly(ADP-ribose) Glycohydrolase
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Identification of an Inhibitor Binding Site of Poly(ADP-ribose) Glycohydrolase

机译：聚（ADP-核糖）糖水解酶抑制剂结合位点的鉴定

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摘要

Polymers of ADP-ribose involved in the maintenance of genomic integrity are converted to free ADP-ribose by the action of poly(ADP-ribose) glycohydrolase (PARG). As an approach to mapping functions of PARG onto the amino acid sequence of the protein, we report here experiments that identify an amino acid residue involved in the binding of potent PARG inhibitors. A photoreactive inhibitor, [alpha-~(32)P]-8-azidoadenosie diphosphate (hydroxymethyl)pyrrolidinediol (8-N_3-ADP-HPD), was used to photolabel a recombinant bovine PARG catalytic fragment (rPARG-CF). N-Terminal sequencing of tryptic and subtilitic peptides of photoderivatized rPARG-CF identified tyrosine 796 (Y796), a residue conserved in PARG across a wide range of organisms, as a site of photoderivatization. Site-directed mutants where this tyrosine residue was replaced with an alanine residue 9Y796A) had a nearly 8-fold decrease in catalytic efficiency (k_(cat)/K_M), while replacement with a tryptophan residue (Y796W) had little effect on catalytic efficiency. Surface plasmon resonance spectroscopy using the PARG inhibitor 8-(aminohexyl)amino-ADP-HPD demonstrated that the binding constant of the inhibitor for Y796A was 21-fold lower (K_D = 170 nM) than that of wild-type of wild-type PARG (K_D = 8.2 nM), while Y796W displayed a binding affinity similar to that of the wild-type enzyme. Our results indicate that Y796 is involved in inhibitor binding to PARG via a ring stacking interaction and identify a highly conserved region o the protein that putatively contains other residues involved in catalytic activity and/or substrate recognition.
机译：通过聚（ADP-核糖）糖水解酶（PARG）的作用将参与基因组完整性维持的ADP-核糖的聚合物转化为游离的ADP-核糖。作为将PARG功能映射到蛋白质氨基酸序列上的一种方法，我们在这里报告鉴定与有效PARG抑制剂结合的氨基酸残基的实验。光反应抑制剂，[α-〜（32）P] -8-叠氮腺苷二磷酸（羟甲基）吡咯烷二醇（8-N_3-ADP-HPD），用于光标记重组牛PARG催化片段（rPARG-CF）。光衍生rPARG-CF的胰蛋白酶和亚脂肽的N端测序确定酪氨酸796（Y796）是光衍生化的一个位点，酪氨酸796（Y796）是PARG在多种生物中的保守残基。酪氨酸残基被丙氨酸残基9Y796A取代的定点突变体的催化效率（k_（cat）/ K_M）降低了近8倍，而色氨酸残基（Y796W）的替代对催化效率的影响很小。使用PARG抑制剂8-（氨基己基）氨基-ADP-HPD的表面等离振子共振光谱表明，抑制剂对Y796A的结合常数比野生型PARG的结合常数低21倍（K_D = 170 nM）。 （K_D = 8.2 nM），而Y796W表现出与野生型酶相似的结合亲和力。我们的结果表明，Y796通过环堆叠相互作用参与了抑制剂与PARG的结合，并确定了该蛋白的高度保守区域，该区域推测含有与催化活性和/或底物识别有关的其他残基。

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