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Catalytic Site Atlas

CSA LITERATURE entry for 1k4t

E.C. nameDNA topoisomerase
SpeciesHomo sapiens (Human)
E.C. Number (IntEnz) 5.99.1.2
CSA Homologues of 1k4tThere are 19 Homologs
CSA Entries With UniProtID P11387
CSA Entries With EC Number 5.99.1.2
PDBe Entry 1k4t
PDBSum Entry 1k4t
MACiE Entry 1k4t

Literature Report

IntroductionEukaryotic DNA topoisomerase I (topo I) is an enzyme that acts to relax supercoils generated during transcription and DNA replication. Due to of the size of the eukaryotic chromosome, removal of these supercoils can only be accomplished locally by introducing breaks into the DNA helix. Topo I mediates DNA relaxation by creating a transient single-strand break in the DNA duplex. This transient nick allows the broken strand to rotate around its intact complement, effectively removing local supercoils. Strand nicking results from the transesterification of an active-site tyrosine (Tyr-723) at a DNA phosphodiester bond forming a 3-phosphotyrosine covalent enzyme-DNA complex. After DNA relaxation, the covalent intermediate is reversed when the released 5-OH of the broken strand reattacks the phosphotyrosine intermediate in a second transesterification reaction. The rate of religation is normally much faster than the rate of cleavage, and this ensures that the steady-state concentration of the covalent 3-phosphotyrosyl topo I DNA complex remains low.
However, a variety of DNA lesions and drugs have been shown to stabilize the covalent 3-phosphotyrosyl intermediate. For example, camptothecin (CPT) is a natural product that was originally discovered because of its antitumor activity and was later demonstrated to cause the accumulation of topo I DNA adducts in vitro and in vivo.CPTs bind the covalent 3-phosphotyrosyl intermediate and specifically block DNA religation, thus converting topo I into a DNA-damaging agent. Topo I is the sole intramolecular target of CPT, and the cytotoxic effects of CPT poisoning are S-phase specific. During DNA replication, the replication fork is thought to collide with the trapped topo I DNA complexes, resulting in double-strand breaks and ultimately cell death.
MechansimThe nucleophilic Tyr723 attacks the backbone posphate group leading to cleavage, the leaving group 5'-oxygen is protonated by Lys532. Controlled rotation of the DNA helix then relaxes its supercoil, after which the nicked strand is re-ligated.

Catalytic Sites for 1k4t

Annotated By Reference To The Literature - Site 1 (Perform Site Search)
ResidueChainNumberUniProtKB NumberFunctional PartFunctionTargetDescription
TyrA723723macie:sideChainTyr attacks the phosphate back bone to form a phosphotyrosine intermediate.
ArgA488488macie:sideChainStabilises transition state by hydrogen bonding to the non-bridging oxygen.
ArgA590590macie:sideChainStabilises transition state by hydrogen bonding to the non-bridging oxygen. Note could also contribute to lowering pKa of Tyr723 leading to its deprotonation for nucleophilic attack.
HisA632632macie:sideChainStabilises transition state. Note was originally thought to eb the general acid.
LysA532532macie:sideChainKnown to act as the general acid which protonates the leaving 5'-oxygen atom.

Literature References

Notes:Note 2005 paper stating Lys532 is an essential catalytic residue.
Stewart L
A model for the mechanism of human topoisomerase I.
Science 1998 279 1534-1541
PubMed: 9488652
Interthal H
The role of lysine 532 in the catalytic mechanism of human topoisomerase I.
J Biol Chem 2004 279 2984-2992
PubMed: 14594810
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