TOPOISOMERASES MAGNESIUM BINDING

PROTIEN:
Topoisomerases are one of the most important DNA-backbone processing enzymes. They are involved in
important biological activities such DNA replication, transcription, and chromosome condensation,
which necessitate enzymes that can regulate the nucleic acid's topological alterations. Their biological,
biophysical, and structural features have all been exhaustively examined, as well as their molecular
mechanism(s) of action. To summaries, topoisomerases cause a topological shift in a DNA chain by
unwinding or supercoiling the double helix, which relieves the torsional strain imposed by DNA
processing. Only a brief break along the DNA phosphodiester backbone, followed by strand passage
through the gate, and lastly the resealing of the backbone break, can accomplish this. The nucleophilic
attack of a tyrosine residue in the catalytic pocket on a phosphodiester link of the nucleic acid backbone
is what causes topoisomerase-mediated cleavage. At the split deoxyribose group, the
transphosphorylation event creates a covalent protein–nucleic acid connection as well as a free hydroxyl
moiety. The topological change is caused by strand passing of a DNA filament via the gate created by
cleavage or by controlled rotation of the cleaved strand around the intact filament. After that, the
original link is resealed by phosphoryl transfer from the tyrosine residue to the broken deoxyribose
hydroxyl. The catalytic cycle is completed when the covalent enzyme–DNA bond is released. Type I
(single-strand break) and type II (dual-strand break) topoisomerases are distinguished by the number of
DNA strands involved in the cleavage reaction (double-strand break). At least one type I and one type II
topoisomerase component can be found in each cellular entity. The amino-acidic sequence and
structure of the proteins, the reaction process, and the sugar position (5′ or 3′) at which the protein
becomes covalently bonded to the nucleic acid through the phosphate group are all considered in
further categorizations.

Typ Subfamily Name Organism(s) ATP Mg Linkage to Mechanism Toprim

e required required tyrosine

I IA Topo I Eubacteria, / + 5′ Strand Yes

archaea, plants passage

Topo Eubacteria, / + 5′ Strand Yes

III archaea, passage
eukaryotes
Topoisomerase IA and II bind covalently to the cleaved DNA's 5′-phosphoryl group, whereas
topoisomerases IB and IC bind to the 3′-phosphoryl group.

For complete catalytic activity, topoisomerases frequently require cofactors. Through binding and
hydrolysis activities, ATP (type II family and reverse gyrase) modulates the conformational changes
required for enzyme function. Divalent metal ions, primarily Mg2+, are extremely significant because, in
addition to working in combination with ATP, they provide structural and catalytic activities. Mg2+ is
required for topoisomerase IA and topoisomerase II to relax supercoiled DNA. A metal ion is not
necessary to participate in the catalytic cycle and does not directly bind the protein in the case of type IB
enzymes. Mg2+, on the other hand, effectively enhances catalytic activity, perhaps making the rate-
limiting DNA-release step at the conclusion of the enzymatic cycle easier 

Mg2+ can help in transphosphorylation in a variety of ways. In fact, it can stabilize the arriving negative
tyrosinated species, neutralize the pentacoordinate negatively charged transition state, and/or assist in
the stability of the negatively charged leaving group using a general base (outer sphere) mechanism.
Despite the fact that a number of experiments have been carried out to replicate nucleic acid hydrolytic
processes, there are currently no accurate quantum mechanical studies on this metal ion-mediated
process. It would be particularly interesting to completely comprehend the reasons that cause
topoisomerases to preferentially attack outer sphere tyrosine over inner sphere water, with the
opposite occurring in a related class of nucleases. Although conserved tyrosine residues are frequently
found in the active sites of DNA nucleases and polymerases (Kienow fragment, T5 flap endonuclease,
staphylococcal nuclease), they do not appear to participate in catalysis and instead contribute in the
creation of ternary complexes. This is an example of how nature can effectively produce different
catalytic effects by enabling enzymes to perform distinct chemical reactions even in the presence of
essentially the same functions at the active site.

Assignment # 2

Hoor Khattak

Biotechnology

3rd Semester