Lynn R M, Wang J C
Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138.
Proteins. 1989;6(3):231-9. doi: 10.1002/prot.340060305.
Tyrosine 319 of E. coli topoisomerase I is shown to be the active site tyrosine that becomes covalently attached to a DNA 5' phosphoryl group during the transient breakage of a DNA internucleotide bond by the enzyme. The tyrosine was mapped by trapping the covalent complex between the DNA and DNA topoisomerase I, digesting the complex exhaustively with trypsin, and sequencing the DNA-linked tryptic peptide. Site-directed mutagenesis converting Tyr-319 to a serine or phenylalanine completely inactivates the enzyme. The structure of the enzyme and its catalysis of DNA strand breakage, passage, and rejoining are discussed in terms of the available information.
大肠杆菌拓扑异构酶I的酪氨酸319被证明是活性位点酪氨酸，在该酶短暂断裂DNA核苷酸间键的过程中，它会与DNA 5'磷酸基团共价结合。通过捕获DNA与DNA拓扑异构酶I之间的共价复合物，用胰蛋白酶彻底消化该复合物，并对与DNA相连的胰蛋白酶肽段进行测序，确定了该酪氨酸的位置。将Tyr-319定点突变为丝氨酸或苯丙氨酸会使该酶完全失活。根据现有信息讨论了该酶的结构及其对DNA链断裂、通过和重新连接的催化作用。
