Structure-function analysis of the mammalian DNA polymerase beta active site: Role of aspartic acid 256, arginine 254, and arginine 258 in nucleotidyl transfer
Structure-function analysis of the mammalian DNA polymerase beta active site: Role of aspartic acid 256, arginine 254, and arginine 258 in nucleotidyl transfer
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DOI：
10.1021/bi00049a008
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发表时间：
1995-12-12
期刊：
影响因子：
2.9
通讯作者：
Hostomska, Z
中科院分区：
文献类型：
--
作者：
Menge, KL;Hostomsky, Z;Hostomska, Z
大鼠DNA聚合酶β的催化结构域的晶体结构显示，Asp 256位于之前确定的活性位点残基Asp 190和Asp 192附近。我们已经制备了野生型和几种突变形式的人和大鼠pol β的核苷酸转移活性，并对其进行了动力学表征。在本文中，我们报告了残基Asp 256和两个相邻残基Arg 254和Arg 258中突变体的K-m（dTTP），K（m）（（dT）16）和k（cat）的稳态动力学测定，所有这些残基都位于pol β结构中β 7链的中心。Asp 256突变为丙氨酸消除了pol β的酶活性。与野生型相比，用谷氨酸（D256 E）保守替换导致k（cat）降低320倍。与野生型相比，用丙氨酸（R254 A）替换Arg 254导致k（cat）降低50倍。D256 E和R254 A的K（m）（（dT）16）相对于野生型增加约18倍。用赖氨酸替换Arg 254导致k（cat）降低15倍，K（m）增加5倍（（dT）16）。这些动力学观察支持Asp 256和Arg 254在定位二价金属离子和底物中的作用，其在精确的几何方向中用于有效的催化。Arg 258突变为丙氨酸（R258 A）导致K-m（dTTP）增加10倍，K（m）（（dT）16）增加65倍，但不导致k（cat）变化。这些观察结果进行了讨论的背景下的三维结构的催化结构域的聚合酶β和三元复合物的聚合酶β，ddCTP，和DNA。
The crystal structure of the catalytic domain of rat DNA polymerase beta revealed that Asp256 is located in proximity to the previously identified active site residues Asp190 and Asp192. We have prepared and kinetically characterized the nucleotidyl transfer activity of wild type and several mutant forms of human and rat pol beta. Herein we report steady-state kinetic determinations of K-m(dTTP), K(m)((dT)16), and k(cat) for mutants in residue Asp256 and two neighboring residues, Arg254 and Arg258, all centrally located on strand beta 7 in the pol beta structure. Mutation of Asp256 to alanine abolished the enzymatic activity of pol beta. Conservative replacement with glutamic acid (D256E) led to a 320-fold reduction of k(cat) compared to wild type. Replacement of Arg254 with an alanine (R254A) resulted in a 50-fold reduction of k(cat) compared to wild type. The K(m)((dT)16) of D256E and R254A increased about 18-fold relative to wild type. Replacement of Arg254 with a lysine resulted in a 15-fold decrease in k(cat), and a 5-fold increase in the K(m)((dT)16). These kinetic observations support a role of Asp256 and Arg254 in the positioning of divalent metal ions and substrates in precise geometrical orientation for efficient catalysis. The mutation of Arg258 to alanine (R258A) resulted in a 10-fold increase in K-m(dTTP) and a 65-fold increase in K(m)((dT)16) but resulted in no change of k(cat). These observations are discussed in the context of the three-dimensional structures of the catalytic domain of pol beta and the ternary complex of pol beta, ddCTP, and DNA.