1XO1

T5 5'-EXONUCLEASE MUTANT K83A

1XO1 の概要

• エントリーDOI: 10.2210/pdb1xo1/pdb
• 分子名称: 5'-EXONUCLEASE (2 entities in total)
• 機能のキーワード: hydrolase, exonuclease, nuclease
• 由来する生物種: Enterobacteria phage T5
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 66867.53

構造登録者

Ceska, T.A.,Suck, D.,Sayers, J.R. (登録日: 1998-11-19, 公開日: 1999-04-02, 最終更新日: 2023-08-23)

主引用文献

Garforth, S.J.,Ceska, T.A.,Suck, D.,Sayers, J.R.
Mutagenesis of conserved lysine residues in bacteriophage T5 5'-3' exonuclease suggests separate mechanisms of endo-and exonucleolytic cleavage.
Proc.Natl.Acad.Sci.USA, 96:38-43, 1999

PubMed Abstract: Efficient cellular DNA replication requires the activity of a 5'-3' exonuclease. These enzymes are able to hydrolyze DNA.DNA and RNA.DNA substrates exonucleolytically, and they are structure-specific endonucleases. The 5'-3' exonucleases are conserved in organisms as diverse as bacteriophage and mammals. Crystal structures of three representative enzymes identify two divalent-metal-binding sites typically separated by 8-10 A. Site-directed mutagenesis was used to investigate the roles of three lysine residues (K83, K196, and K215) situated near two metal-binding sites in bacteriophage T5 5'-3' exonuclease. Neither K196 nor K215 was essential for either the exo- or the endonuclease activity, but mutation of these residues increased the dissociation constant for the substrate from 5 nM to 200 nM (K196A) and 50 nM (K215A). Biochemical analysis demonstrated that K83 is absolutely required for exonucleolytic activity on single-stranded DNA but is not required for endonucleolytic cleavage of flap structures. Structural analysis of this mutant by x-ray crystallography showed no significant perturbations around the metal-binding sites in the active site. The wild-type protein has different pH optima for endonuclease and exonuclease activities. Taken together, these results suggest that different mechanisms for endo- and exonucleolytic hydrolysis are used by this multifunctional enzyme.

PubMed: 9874768
DOI: 10.1073/pnas.96.1.38

実験手法

X-RAY DIFFRACTION (2.5 Å)