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Catalytic Site Atlas

CSA LITERATURE entry for 1j53

E.C. nameDNA-directed DNA polymerase
SpeciesEscherichia coli (Bacteria)
E.C. Number (IntEnz) 2.7.7.7
CSA Homologues of 1j53
CSA Entries With UniProtID P03007
CSA Entries With EC Number 2.7.7.7
PDBe Entry 1j53
PDBSum Entry 1j53
MACiE Entry 1j53

Literature Report

IntroductionEscherichia coli replicative DNA polymerase III, a member of family C of DNA polymerases, serves as the main DNA polymerase for E. coli. The polymerase activity is catalysed by the alpha subunit, with the epsilon subunit catalysing the 3'-5' exonuclease proofreading activity. The N-terminal domain of the epsilon subunit is the archetypal member of the DnaQ superfamily of 3'-5' exonucleases.
MechansimThe active site contains two metal ions (A and B), which are likely to be magnesium in the native enzyme, but manganese can also be accommodated. They are coordinated to conserved acidic residues: Asp 12, Glu 14 and Asp 167. Asp 103 binds two fixed water molecules that also coordinate Metal B. The overall reaction is back-side attack of the water nucleophile on the phosphate linking the terminal nucleoside to the rest of the DNA molecule, shortening the newly replicated DNA by one base - the mechanism is common to two-metal 3'-5' exonucleases.
The proposed mechanism is:
1) Metal A activates water as nucleophile. His 162, made more basic by hydrogen bonding to Glu 61, deprotonates water as water (as hydroxide) attacks the phosphate of the scissile phosphoester bond. This phosphate group is made more electrophilic by coordination to Metal B.
2) This produces a pentacovalent phosphorane intermediate. Metal B stabilises the negative charge on the phosphate group in the intermediate and the transition states. Glu 14 deprotonates the phosphorane (abstracting the proton from what was the attacking hydroxide) triggering collapse of the intermediate. Metal B coordinates the 3' phosphate oxygen of the substrate, assisting it to leave.
3) The products of the reaction are a dNMP and the shortened DNA molecule.
Reaction

Catalytic Sites for 1j53

Annotated By Reference To The Literature - Site 1 (Perform Site Search)
ResidueChainNumberUniProtKB NumberFunctional PartFunctionTargetDescription
GluA6161macie:sideChainGlu 61 hydrogen bonds to His 162; this may increase the basicity of His 162.
GluA1414macie:sideChainGlu 14 coordinates Metal A and may orient the attacking hydroxide. Glu 14 may also have a role in deprotonating the phosphorane intermediate, triggering collapse and product formation.
HisA162162macie:sideChainHis 162 acts as a general base to deprotonate water, facilitating effective attack of hydroxide.

Literature References

Notes:The catalytic importance of Glu 61 is not well established. Glu 61 is not conserved, but may account for the different rates of hydrolysis for the four dNMPs that is not observed for other exonucleases. The general base role of Glu 14 is also not well established. The mechanism can also conceivably proceed in a single step, with the pentacovalent structure occurring during the single transition state rather than an intermediate.
Hamdan S
Structural basis for proofreading during replication of the Escherichia coli chromosome.
Structure 2002 10 535-546
PubMed: 11937058
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