Lynn R M, Bjornsti M A, Caron P R, Wang J C
Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA 02138.
Proc Natl Acad Sci U S A. 1989 May;86(10):3559-63. doi: 10.1073/pnas.86.10.3559.
Extensive digestion of the covalent intermediate between DNA and Saccharomyces cerevisiae DNA topoisomerase I with trypsin yields a 7-amino acid peptide covalently linked to DNA. Direct sequencing of the DNA-linked peptide identifies Tyr-727 as the active site tyrosine that forms an O4-phosphotyrosine bond with DNA when the enzyme cleaves a DNA phosphodiester bond. Site-directed mutagenesis of the cloned yeast TOP1 gene encoding the enzyme confirms the essentiality of Tyr-727 for the relaxation of supercoiled DNA by the enzyme. From amino acid sequence homology, Tyr-771 and -773 are readily identified as the active site tyrosines of Schizosaccharomyces pombe and human DNA topoisomerase I, respectively. Sequence comparison and site-directed mutagenesis also implicate Tyr-274 of vaccinia virus DNA topoisomerase as the active site residue. There appears to be a 70-amino acid domain near the carboxyl terminus of eukaryotic DNA topoisomerase I and vaccinia topoisomerase, within which the active site tyrosine resides.
用胰蛋白酶充分消化DNA与酿酒酵母DNA拓扑异构酶I之间的共价中间体，可产生一个与DNA共价连接的7氨基酸肽。对与DNA相连的肽进行直接测序，确定Tyr-727为活性位点酪氨酸，当该酶切割DNA磷酸二酯键时，它与DNA形成O4-磷酸酪氨酸键。对编码该酶的克隆酵母TOP1基因进行定点诱变，证实了Tyr-727对于该酶松弛超螺旋DNA的必要性。根据氨基酸序列同源性，分别容易确定Tyr-771和Tyr-773为粟酒裂殖酵母和人DNA拓扑异构酶I的活性位点酪氨酸。序列比较和定点诱变也表明痘苗病毒DNA拓扑异构酶的Tyr-274为活性位点残基。真核DNA拓扑异构酶I和痘苗拓扑异构酶的羧基末端附近似乎有一个70氨基酸结构域，活性位点酪氨酸位于其中。
