The active site for E. coli   topoisomerase I is located in the amino-terminal fragment.  The catalytic tyrosine (Tyr319) is located on domain II at the interface made by domains I, III, and IV.  Tyrosine 319 interacts with several highly conserved amino acids and water molecules to form an extensive network of hydrogen bonds.  A second shell of conserved amino acids surrounds the active site tyrosine.   Three acidic residues are near the active site and are very similar to the acidic residues that coordinate two divalent cations in the exonuclease catalytic site of Klenow fragment although topoisomerase mutational studies showed that only the Glu-9 and Arg-321 affect transesterification between the active site tyrosine and the scissile DNA phosphorous. The architecture of the enzyme suggests that domains II and III can be lifted off domains I and IV as a rigid body, using the strands joining domains II and IV as a hinge (Lima, 1994). The cleavage step of the reaction involves a covalent bond between Tyr 319 and the 5'-phosphoryl end of the broken strand. The 3' end of the broken DNA strand is likely to remain associated with either domain I or IV. A proton is removed from the tyrosyl hydroxyl in the formation of the phosphotyrosine bond, and a proton is added to the deoxyribose 3' oxygen as it departs from the scissile phosphate. When the DNA rejoins the steps are reversed. Glu-9 is thought to be the best candidate for both the proton donor and acceptor (Chen, 1998).

Back to Index Page Back to the description of the active site and catalytic strategy of topoisomerase I

Updated:  December 11, 1998

Comments, Questions: luebkeac@uwec.edu