Sand box 211: Difference between revisions

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<scene name='Sand_box_211/Lys215216/2'>Lys215 and Arg216</scene> form part of a helix–loop–helix feature. <scene name='Sand_box_211/Arg33/2'>Arg33</scene> binds to a phosphodiester residue in the 3'  end of the cleavage site.
<scene name='Sand_box_211/Lys215216/2'>Lys215 and Arg216</scene> form part of a helix–loop–helix feature. <scene name='Sand_box_211/Arg33/2'>Arg33</scene> binds to a phosphodiester residue in the 3'  end of the cleavage site.


[[Image:mg.jpg | thumb | left | The metal ions' interaction]]The reaction only takes place if at least two divalent metal ions are bound to the enzyme <ref>PMID:18697748</ref>. However, the DNA binding doesn't need the presence of metal ions. Metal ions participate in the enzymatic catalysis of phosphodiester bond in several ways. For example, they can act as a nucleophile or a general base. They also participate in the catalysis of phosphate diester hydrolysis reactions by interacting with the oxygens which are not involved in the scissile phosphate (figure left).
[[Image:mg.jpg | thumb | left | The metal ions' interaction]]The reaction only takes place if at least two divalent metal ions are bound to the enzyme. However, the DNA binding doesn't need the presence of metal ions. Metal ions participate in the enzymatic catalysis of phosphodiester bond in several ways. For example, they can act as a nucleophile or a general base. They also participate in the catalysis of phosphate diester hydrolysis reactions by interacting with the oxygens which are not involved in the scissile phosphate (figure left).




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<references/>
<references/>
1.↑ Ceska TA, Sayers JR, Stier G, Suck D. A helical arch allowing single-stranded DNA to thread through T5 5'-exonuclease. Nature. 1996 Jul 4;382(6586):90-3. PMID:8657312[http://www.ncbi.nlm.nih.gov/pubmed/8657312?dopt=Abstract]doi:10.1038/382090a0 [http://dx.doi.org/10.1038/382090a0]
2.↑ Ceska TA, Suck D, Sayers JR. 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 Jan 5;96(1):38-43. PMID:9874768[http://www.ncbi.nlm.nih.gov/pubmed/9874768?dopt=Abstract]doi:10.1073/pnas.96.1.38 [http://dx.doi.org/10.1073/pnas.96.1.38]
3.↑ Mark R.Tock, Elaine Frary, Jon R.Sayers and Jane A.Grasby. Dynamic evidence for metal ion catalysis in the reaction mediated by a flap endonuclease. The EMBO Journal 2003 Mar 3;22(5):995-1004. PMID:12606565 [http://www.ncbi.nlm.nih.gov/pubmed/12606565] doi:10.1093/emboj/cdg098 [http://dx.doi.org/10.1093/emboj/cdg098]
4.↑ Karl Syson, Christopher Tomlinson, Brian R. Chapados, Jon R. Sayers, John A. Tainer, Nicholas H. Williams and Jane A. Grasby. Three Metal Ions Participate in the Reaction Catalyzed by T5 Flap Endonuclease. J Biol Chem. 2008 October 17; 283(42): 28741–28746. PMID:18697748 [http://www.ncbi.nlm.nih.gov/pubmed/18697748] doi:10.1074/jbc.M801264200 [http://dx.doi.org/10.1074/jbc.M801264200]
    
    
5.↑ Dervan JJ, Feng M, Patel D, Grasby JA, Artymiuk PJ, Ceska TA, Sayers JR. Interactions of mutant and wild-type flap endonucleases with oligonucleotide substrates suggest an alternative model of DNA binding. Proc Natl Acad Sci U S A. 2002 Jun 25;99(13):8542-7 PMID:12084915 [http://www.ncbi.nlm.nih.gov/pubmed/12084915] doi:10.1073/pnas.082241699 [http://dx.doi.org/10.1073/pnas.082241699]   
5.↑ Dervan JJ, Feng M, Patel D, Grasby JA, Artymiuk PJ, Ceska TA, Sayers JR. Interactions of mutant and wild-type flap endonucleases with oligonucleotide substrates suggest an alternative model of DNA binding. Proc Natl Acad Sci U S A. 2002 Jun 25;99(13):8542-7 PMID:12084915 [http://www.ncbi.nlm.nih.gov/pubmed/12084915] doi:10.1073/pnas.082241699 [http://dx.doi.org/10.1073/pnas.082241699]   
6.↑ Pickering TJ, Garforth S, Sayers JR, Grasby JA. Variation in the steady state kinetic parameters of wild type and mutant T5 5'-3'-exonuclease with pH. Protonation of Lys-83 is critical for DNA binding. J Biol Chem. 1999 Jun 18;274(25):17711-7. PMID:10364212 [http://www.ncbi.nlm.nih.gov/pubmed/10364212] doi:10.1074/jbc.274.25.17711 [http://dx.doi.org/10.1074/jbc.274.25.17711]
7.↑ Garforth SJ, Patel D, Feng M, Sayers JR. Unusually wide co-factor tolerance in a metalloenzyme; divalent metal ions modulate endo-exonuclease activity in T5 exonuclease. Nucleic Acids Res. 2001 Jul 1;29(13):2772-9. PMID:11433022 [http://www.ncbi.nlm.nih.gov/pubmed/11433022] doi:10.1093/nar/29.13.2772 [http://dx.doi.org/10.1093/nar/29.13.2772]


8.↑ Sengerová B, Tomlinson C, Atack JM, Williams R, Sayers JR, Williams NH, Grasby JA. Brønsted analysis and rate-limiting steps for the T5 flap endonuclease catalyzed hydrolysis of exonucleolytic substrates. Biochemistry. 2010 Sep 21;49(37):8085-93. PMID: 20698567 doi:10.1021/bi100895j [http://dx.doi.org/10.1021/bi100895j]
8.↑ Sengerová B, Tomlinson C, Atack JM, Williams R, Sayers JR, Williams NH, Grasby JA. Brønsted analysis and rate-limiting steps for the T5 flap endonuclease catalyzed hydrolysis of exonucleolytic substrates. Biochemistry. 2010 Sep 21;49(37):8085-93. PMID: 20698567 doi:10.1021/bi100895j [http://dx.doi.org/10.1021/bi100895j]
9.↑ Tomlinson CG, Syson K, Sengerová B, Atack JM, Sayers JR, Swanson L, Tainer JA, Williams NH, Grasby JA. Neutralizing mutations of carboxylates that bind metal 2 in T5 flap endonuclease result in an enzyme that still requires two metal ions. J Biol Chem. 2011 Sep 2;286(35):30878-87. Epub 2011 Jul 6. PMID: 21734257 doi:10.1074/jbc.M111.230391 [http://dx.doi.org/10.1074/jbc.M111.230391]


10.↑ Pickering TJ, Garforth SJ, Thorpe SJ, Sayers JR, Grasby JA. A single cleavage assay for T5 5'-->3' exonuclease: determination of the catalytic parameters forwild-type and mutant proteins. Nucleic Acids Res. 1999 Feb 1;27(3):730-5. PMID: 9889266 doi:10.1093/nar/27.3.730 [http://dx.doi.org/10.1093/nar/27.3.730]
10.↑ Pickering TJ, Garforth SJ, Thorpe SJ, Sayers JR, Grasby JA. A single cleavage assay for T5 5'-->3' exonuclease: determination of the catalytic parameters forwild-type and mutant proteins. Nucleic Acids Res. 1999 Feb 1;27(3):730-5. PMID: 9889266 doi:10.1093/nar/27.3.730 [http://dx.doi.org/10.1093/nar/27.3.730]
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