Biochemical characterization of an invariant histidine involved in Escherichia coli DNA topoisomerase I catalysis
Biochemical characterization of an invariant histidine involved in Escherichia coli DNA topoisomerase I catalysis
复制标题
DOI：
10.1074/jbc.m112019200
复制
发表时间：
2002-04-12
影响因子：
4.8
通讯作者：
Mondragón, A
中科院分区：
文献类型：
--
作者：
Perry, K;Mondragón, A
在大肠杆菌DNA拓扑异构酶I中，一个不变的组氨酸残基His-365位于IA型DNA拓扑异构酶的活性部位和酪氨酸活性部位附近。考察了其参与DNA弛豫多步催化过程的能力。将His-365突变为丙氨酸、精氨酸、天冬氨酸、天冬氨酸、谷氨酸和谷氨酰胺，以研究其参与一般酸碱催化和结合DNA的能力。对突变体进行了pH依赖的DNA松弛和切割、盐依赖的DNA松弛和盐依赖的DNA结合亲和力的检测。突变体在低盐浓度下以一种pH依赖的方式松弛DNA。所有突变体的pH依赖性与野生型不同，提示His-365与该酶的pH依赖性有关。此外，野生型酶表现出pH依赖的寡肽切割，而H365Q和H365A的切割是不依赖于pH的。H365Q切割DNA的速度与野生型相似，而H365A的DNA切割速度慢于野生型，但总体上可以切割更多的底物。H365A的DNA结合亲和力也低于野生型。在不同的盐浓度下测定了结合亲和力，结果表明，丙氨酸突变体与DNA结合的电荷比拓扑异构酶I的失活形式少半个电荷。这些观察表明，His-365参与了DNA结合，并在生理pH下起到最佳催化作用。
An invariant histidine residue, His-365 in Escherichia coli DNA topoisomerase I, is located at the active site of type IA DNA topoisomerases and near the active site tyrosine. Its ability to participate in the multistep catalytic process of DNA relaxation was investigated. His-365 was mutated to alanine, arginine, asparagine, aspartate, glutamate, and glutamine to study its ability to participate in general acid/base catalysis and bind DNA. The mutants were examined for pH-dependent DNA relaxation and cleavage, salt-dependent DNA relaxation, and salt-dependent DNA binding affinity. The mutants relax DNA in a pH-dependent manner and at low salt concentrations. The pH dependence of all mutants is different from the wild type, suggesting that His-365 is responsible for the pH dependence of the enzyme. Additionally, whereas the wild type enzyme shows pH-dependent oligonuelcotide cleavage, cleavage by both H365Q and H365A is pH-independent. H365Q cleaves DNA with rates similar to the wild type enzyme, whereas H365A has a slower rate of DNA cleavage than the wild type but can cleave more substrate overall. H365A also has a lower DNA binding affinity than the wild type enzyme. The binding affinity was determined at different salt concentrations, showing that the alanine mutant displaces half a charge less upon binding DNA than an inactive form of topoisomerase I. These observations indicate that His-365 participates in DNA binding and is responsible for optimal catalysis at physiological pH.