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Catalytic Site Atlas

CSA LITERATURE entry for 1exn

E.C. nameexodeoxyribonuclease (lambda-induced)
SpeciesBacteriophage t5 ()
E.C. Number (IntEnz)
CSA Homologues of 1exnThere are 15 Homologs
CSA Entries With UniProtID P06229
CSA Entries With EC Number
PDBe Entry 1exn
PDBSum Entry 1exn
MACiE Entry 1exn

Literature Report

IntroductionThe 5'-3' exonuclease enzymes hydrolyse DNA.DNA and RNA.DNA substrates in a structurally specific manner. These enzymes are necessary for efficient cellular DNA replication, with the uncatalysed reaction having a half life of 30 million years at room temperature. The enzymes enhance the rate by 10^15-10^17, allowing the reaction to proceed on a biologically useful time scale.
MechansimThere are several proposed mechanisms for the action of 5'-3' exonuclease, taking into account many possible mechanisms involving up to three catalytic divalent metal ions.
The most accepted mechanism implies the involvement of two catalytic Mg(2+) ions. A water molecule is deprotonated by the conserved basic Lys 83, which remains deprotonated at pH 6 through interactions with a positively charged dicationic metal and the positively charged guanidinium group of Arg 86 (also a conserved residue). This water molecule then acts as a nucleophile towards the scissile phosphate. The pentavalent intermediate is stabilised by a second dicationic Mg while the scissile phosphate oxygen is coordinated to both metal centres. The collapse of the intermediate and reprotonation of the 3' leaving group from the Lys 83 gives the hydrolysis products.
Crystallographic evidence suggests a two-metal-ion catalytic process in homologous enzymes, although this pdb doesn't show the Mg atoms implicated. Later kinetic evidence suggests the presence of three metal cations is necessary. It has also been proposed on evidence from mutagenesis reactions that the endo- and exonucleolytic cleavage mechanisms are distinct.

Catalytic Sites for 1exn

Annotated By Reference To The Literature - Site 1 (Perform Site Search)
ResidueChainNumberUniProtKB NumberFunctional PartFunctionTargetDescription
LysA8383macie:sideChainThe residue acts as a general base towards a water molecule, activating it for nucleophilic attack at the scissile phosphate. Its pKa is modified by interactions with the guanidinium group of Arg 86 and the positive charge of a dicationic Mg ion. The resulting protonated residue then acts as a general acid to the 3' leaving group.
ArgA8686macie:sideChainThe residue's side chain influences the pKa of the general base Lys 83, enhancing its ability to remain deprotonated at relatively low pH levels.

Literature References

Garforth SJ
Mutagenesis of conserved lysine residues in bacteriophage T5 5'-3' exonuclease suggests separate mechanisms of endo-and exonucleolytic cleavage.
Proc Natl Acad Sci U S A 1999 96 38-43
PubMed: 9874768
Yang W
Making and breaking nucleic acids: two-Mg2+-ion catalysis and substrate specificity.
Mol Cell 2006 22 5-13
PubMed: 16600865
Syson K
Three metal ions participate in the reaction catalyzed by T5 flap endonuclease.
J Biol Chem 2008 283 28741-28746
PubMed: 18697748