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首页> 外文期刊>Journal of Molecular Biology >A Conserved Tyrosine Residue Aids Ternary Complex Formation, but not Catalysis, in Phage T5 Flap Endonuclease.
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A Conserved Tyrosine Residue Aids Ternary Complex Formation, but not Catalysis, in Phage T5 Flap Endonuclease.

机译：保守的酪氨酸残基有助于噬菌体T5瓣内切核酸酶中三元复合物的形成，但不催化。

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摘要

The flap endonucleases, or 5' nucleases, are involved in DNA replication and repair. They possess both 5'-3' exonucleolytic activity and the ability to cleave bifurcated, or branched DNA, in an endonucleolytic, structure-specific manner. These enzymes share a great degree of structural and sequence similarity. Conserved acidic amino acids, whose primary role appears to be chelation of essential divalent cation cofactors, lie at the base of the active site. A loop, or helical archway, is located above the active site. A conserved tyrosine residue lies at the base of the archway in phage T5 flap endonuclease. This residue is conserved in the structures of all flap endonucleases analysed to date. We mutated the tyrosine 82 codon in the cloned T5 5' nuclease to one encoding phenylalanine. Detailed analysis of the purified Y82F protein revealed only a modest (3.5-fold) decrease in binding affinity for DNA compared with wild-type in the absence of cofactor. The modified nuclease retains both structure-specific endonuclease and exonuclease activities. Kinetic analysis was performed using a newly developed single-cleavage assay based on hydrolysis of a fluorescently labelled oligonucleotide substrate. Substrate and products were resolved by denaturing HPLC. Steady-state kinetic analysis revealed that loss of the tyrosine hydroxyl function did not significantly impair k(cat). Pre-steady state analysis under single-turnover conditions also demonstrated little change in the rate of reaction compared to the wild-type protein. The pH dependence of the kinetic parameters for the Y82F enzyme-catalysed reaction was bell-shaped as for the wild-type protein. Thus, Y82 does not play a role in catalysis. However, steady-state analysis did detect a large ( reverse similar300-fold) defect in K(M). These results imply that this conserved tyrosine plays a key role in ternary complex formation (protein-DNA-metal ion), a prerequisite for catalysis.
机译：皮瓣内切核酸酶或5'核酸酶参与DNA复制和修复。它们既具有5'-3'的核酸外切酶活性，又具有以核酸内切酶，结构特异性的方式裂解分支或分支的DNA的能力。这些酶具有高度的结构和序列相似性。保守的酸性氨基酸，其主要作用似乎是必需的二价阳离子辅因子的螯合，位于活性位点的基础上。环形或螺旋拱门位于活动站点上方。保守的酪氨酸残基位于噬菌体T5瓣内切核酸酶的牌坊底部。迄今为止，该残基在所有瓣状核酸内切酶的结构中均保守。我们将克隆的T5 5'核酸酶中的酪氨酸82密码子突变为一种编码苯丙氨酸的密码子。对纯化的Y82F蛋白的详细分析显示，与不存在辅因子的野生型相比，与DNA的结合亲和力仅适度降低（3.5倍）。修饰的核酸酶保留了结构特异性核酸内切酶和核酸外切酶的活性。使用新开发的基于荧光标记的寡核苷酸底物水解的单裂解测定法进行动力学分析。通过变性HPLC分离底物和产物。稳态动力学分析表明酪氨酸羟基功能的丧失不会显着损害k（cat）。与野生型蛋白相比，单周转条件下的稳态前分析也显示出反应速率几乎没有变化。与野生型蛋白一样，Y82F酶催化反应动力学参数的pH依赖性呈钟形。因此，Y82在催化中不起作用。但是，稳态分析的确在K（M）中检测到较大的（反向相似的300倍）缺陷。这些结果表明，这种保守的酪氨酸在三元络合物的形成（蛋白质-DNA-金属离子）中起着关键作用，而三元络合物的形成是催化的先决条件。

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