Tse-Dinh Y C
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla 10595.
J Biol Chem. 1991 Aug 5;266(22):14317-20.
Escherichia coli DNA topoisomerase I catalyzes relaxation of negatively supercoiled DNA. The reaction proceeds through a covalent intermediate, the cleavable complex, in which the DNA is cleaved and the enzyme is linked to the DNA via a phosphotyrosine linkage. Each molecule of E. coli DNA topoisomerase I has been shown to have three tightly bound zinc(II) ions required for relaxation activity (Tse-Dinh, Y.-C., and Beran-Steed, R.K. (1988) J. Biol. Chem. 263, 15857-15859). It is shown here that Cd(II) could replace Zn(II) in reconstitution of active enzyme from apoprotein. The role of metal was analyzed by studying the partial reactions. The apoenzyme was deficient in sodium dodecyl sulfate-induced cleavage of supercoiled PM2 phage DNA. Formation of covalent complex with linear single-stranded DNA was also reduced in the absence of metal. However, the cleavage of small oligonucleotide was not affected, and the apoenzyme could religate the covalently bound oligonucleotide to another DNA molecule. Assay of noncovalent complex formation by retention of 5'-labeled DNA on filters showed that the apoenzyme was not inhibited in noncovalent binding to DNA. It is proposed that zinc(II) coordination in E. coli DNA topoisomerase I is required for the transition of the noncovalent complex with DNA to the cleavable state.
大肠杆菌DNA拓扑异构酶I催化负超螺旋DNA的松弛。该反应通过共价中间体，即可裂解复合物进行，其中DNA被裂解，酶通过磷酸酪氨酸键与DNA相连。已证明大肠杆菌DNA拓扑异构酶I的每个分子都有三个紧密结合的锌（II）离子，这是松弛活性所必需的（谢丁，Y.-C.，和贝兰-斯蒂德，R.K.（1988年）《生物化学杂志》263，15857-15859）。本文表明，在从脱辅基蛋白重构活性酶的过程中，镉（II）可以取代锌（II）。通过研究部分反应分析了金属的作用。脱辅基酶在十二烷基硫酸钠诱导的超螺旋PM2噬菌体DNA裂解方面存在缺陷。在没有金属的情况下，与线性单链DNA形成共价复合物的能力也降低了。然而，小寡核苷酸的裂解不受影响，脱辅基酶可以将共价结合的寡核苷酸重新连接到另一个DNA分子上。通过在滤膜上保留5'-标记的DNA来测定非共价复合物的形成，结果表明脱辅基酶在与DNA的非共价结合中没有受到抑制。有人提出，大肠杆菌DNA拓扑异构酶I中的锌（II）配位是使与DNA的非共价复合物转变为可裂解状态所必需的。
