Nucleophile activation in PD...(D/E)xK metallonucleases: an experimental and computational pK(a) study.
Nucleophile activation in PD...(D/E)xK metallonucleases: an experimental and computational pK(a) study.
复制标题
DOI：
10.1016/j.jinorgbio.2010.02.008
复制
发表时间：
2010-06
影响因子：
3.9
通讯作者：
Dupureur, Cynthia M.
中科院分区：
文献类型：
--
作者：
Xie, Fuqian;Briggs, James M.;Dupureur, Cynthia M.
金属核酸酶进行依赖金属的核酸水解。虽然金属离子在这些酶中发挥着多种机制作用，但由于缺乏明显的通用碱基，攻击水是如何被激活的仍不清楚。所有的争论都取决于与该物种非常接近的活性位点部分的适当pKa，而对特定水分子的pKa的测量很难通过实验获得。本文描述了一种计算方法，用于探索具有共同PD…（D/E）xK基序的金属核酸酶中水衍生亲核试剂的局部静电影响。我们利用UHBD来预测活性位点基团的pKa，包括定位为亲核试剂的水分子的pKa。在Mg(II)-PvuII酶配合物中，一个Mg(II)-连接的水分子氢键与保守的Lys70的pKa计算为6.5。在单独的实验中去除金属和Lys基团上的电荷；两者都导致了该水分子pKa的升高，这与这两个部分对降低pKa的贡献一致。这种行为在其它PD…（D/E）xK金属核酸酶中保持不变。根据pH值对单次周转率常数的依赖关系提取的pKa值与以往的实验数据进行了比较，并对pKa值进行了预测。
Metallonucleases conduct metal dependent nucleic acid hydrolysis. While metal ions serve in multiple mechanistic capacities in these enzymes, precisely how the attacking water is activated remains unclear for those lacking an obvious general base. All arguments hinge on appropriate pKa’s for active site moieties very close to this species, and measurement of the pKa of a specific water molecule is difficult to access experimentally. Here we describe a computational approach for exploring the local electrostatic influences on the water-derived nucleophile in metallonucleases featuring the common PD…(D/E)xK motif. We utilized UHBD to predict the pKa’s of active site groups, including that of a water molecule positioned to act as a nucleophile. The pKa of a Mg(II)-ligated water molecule hydrogen bonded to the conserved Lys70 in a Mg(II)-PvuII enzyme complex was calculated to be 6.5. The metal and the charge on the Lys group were removed in separate experiments; both resulted in the elevation of the pKa of this water molecule, consistent with contributions from both moieties to lowering this pKa. This behavior is preserved among other PD…(D/E)xK metallonucleases. pKa’s extracted from the pH dependence of the single turnover rate constant are compared to previous experimental data and the above predicted pKa’s.
登录
查看更多内容
影响因子：
2.9
作者：
Antosiewicz, J;McCammon, JA;Gilson, MK
通讯作者：
Gilson, MK
DOI：
10.1038/79032
发表时间：
2000-09-01
期刊：
NATURE STRUCTURAL BIOLOGY
影响因子：
--
作者：
Deibert, M;Grazulis, S;Huber, R
通讯作者：
Huber, R
影响因子：
5.6
作者：
ANTOSIEWICZ, J;MCCAMMON, JA;GILSON, MK
通讯作者：
GILSON, MK
影响因子：
2.9
作者：
Haq, I;O'Brien, R;Ladbury, JE
通讯作者：
Ladbury, JE
影响因子：
15
作者：
Horton, NC;Connolly, BA;Perona, JJ
通讯作者：
Perona, JJ