Sand box 211: Difference between revisions

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<scene name='Sand_box_211/Sheet/1'>beta-sheets</scene>. These form the base of the active site pocket which bind the catalytic metal ions.

The active site possesses 8 conserved <scene name='Sand_box_211/8_residus/4'>acidic residues</scene> (Asp26, Asp68, Glu128, Asp130, Asp153, Asp155, Asp201, Asp204) which interact with divalent metal ions. <scene name='Sand_box_211/Try82/3'>Tyr82</scene> is also a conserved residue located in the active site, but it doesn't seem to have an important role since its mutation doesn't dramatically change the affinity to bind DNA.

The active site possesses 8 conserved <scene name='Sand_box_211/8_residus/4'>acidic residues</scene> (Asp26, Asp68, Glu128, Asp130, Asp153, Asp155, Asp201, Asp204) which interact with divalent metal ions. <scene name='Sand_box_211/Try82/3'>Tyr82</scene> is also a conserved residue located in the active site, but it doesn't seem to have an important role since its mutation doesn't dramatically change the affinity to bind DNA <ref>PMID:12126622</ref>.

<scene name='Sand_box_211/6residus/3'>Six residues</scene> (Arg33, Lys83, Arg172, Lys196, Lys215, Arg216 and Lys241) near the active site permit binding to branched DNA.

<scene name='Sand_box_211/Lys83/3'>Lys83</scene> is positioned in the helical arch region close to metal site 1. It has an important binding role as well as a catalytic role. It was shown that DNA binding is pH dependent which means that the T5 5'-exonuclease requires protonation of Lys83 to be able to bind to DNA. The mechanism of the Lys83 in the catalytic activity is still unknown, but it has been proposed that Lys83 acts as a general base/acid activating water to attack the scissile phosphodiester bond and protonating the leaving oxygen <ref>PMID:10364212</ref>.

<scene name='Sand_box_211/Lys83/3'>Lys83</scene> is positioned in the helical arch region close to metal site 1. It has an important binding role as well as a catalytic role <ref>PMID: 20698567</ref>. It was shown that DNA binding is pH dependent which means that the T5 5'-exonuclease requires protonation of Lys83 to be able to bind to DNA. The mechanism of the Lys83 in the catalytic activity is still unknown, but it has been proposed that Lys83 acts as a general base/acid activating water to attack the scissile phosphodiester bond and protonating the leaving oxygen <ref>PMID:10364212</ref> <ref>PMID:9889266</ref>.

<scene name='Sand_box_211/Lys196/3'>Lys196</scene> is positioned between two metal sites. Its mutation perturbs metal ion binding<ref>PMID:9874768</ref>.

<scene name='Sand_box_211/Lys215arg216lys241/3'>Lys215, Arg216 and Lys241</scene> are important for binding to the 5' overhanging hairpin substrate. Furthermore, residues

<scene name='Sand_box_211/Lys215arg216lys241/3'>Lys215, Arg216 and Lys241</scene> are important for binding to the 5' overhanging hairpin substrate <ref>PMID:12084915</ref>. Furthermore, residues <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. 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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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]

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]

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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@@ Line 26: / Line 26: @@
 <scene name='Sand_box_211/Sheet/1'>beta-sheets</scene>. These form the base of the active site pocket which bind the catalytic metal ions.
-The active site possesses 8 conserved <scene name='Sand_box_211/8_residus/4'>acidic residues</scene> (Asp26, Asp68, Glu128, Asp130, Asp153, Asp155, Asp201, Asp204) which interact with divalent metal ions. <scene name='Sand_box_211/Try82/3'>Tyr82</scene> is also a conserved residue located in the active site, but it doesn't seem to have an important role since its mutation doesn't dramatically change the affinity to bind DNA.
+The active site possesses 8 conserved <scene name='Sand_box_211/8_residus/4'>acidic residues</scene> (Asp26, Asp68, Glu128, Asp130, Asp153, Asp155, Asp201, Asp204) which interact with divalent metal ions. <scene name='Sand_box_211/Try82/3'>Tyr82</scene> is also a conserved residue located in the active site, but it doesn't seem to have an important role since its mutation doesn't dramatically change the affinity to bind DNA <ref>PMID:12126622</ref>.
 <scene name='Sand_box_211/6residus/3'>Six residues</scene> (Arg33, Lys83, Arg172, Lys196, Lys215, Arg216 and Lys241) near the active site permit binding to branched DNA.
-<scene name='Sand_box_211/Lys83/3'>Lys83</scene> is positioned in the helical arch region close to metal site 1. It has an important binding role as well as a catalytic role. It was shown that DNA binding is pH dependent which means that the T5 5'-exonuclease requires protonation of Lys83 to be able to bind to DNA. The mechanism of the Lys83 in the catalytic activity is still unknown, but it has been proposed that Lys83 acts as a general base/acid activating water to attack the scissile phosphodiester bond and protonating the leaving oxygen <ref>PMID:10364212</ref>.
+<scene name='Sand_box_211/Lys83/3'>Lys83</scene> is positioned in the helical arch region close to metal site 1. It has an important binding role as well as a catalytic role <ref>PMID: 20698567</ref>. It was shown that DNA binding is pH dependent which means that the T5 5'-exonuclease requires protonation of Lys83 to be able to bind to DNA. The mechanism of the Lys83 in the catalytic activity is still unknown, but it has been proposed that Lys83 acts as a general base/acid activating water to attack the scissile phosphodiester bond and protonating the leaving oxygen <ref>PMID:10364212</ref> <ref>PMID:9889266</ref>.
 <scene name='Sand_box_211/Lys196/3'>Lys196</scene> is positioned between two metal sites. Its mutation perturbs metal ion binding<ref>PMID:9874768</ref>.
-<scene name='Sand_box_211/Lys215arg216lys241/3'>Lys215, Arg216 and Lys241</scene> are important for binding to the 5' overhanging hairpin substrate. Furthermore, residues
+<scene name='Sand_box_211/Lys215arg216lys241/3'>Lys215, Arg216 and Lys241</scene> are important for binding to the 5' overhanging hairpin substrate <ref>PMID:12084915</ref>. Furthermore, residues <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. 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).
@@ Line 60: / Line 59: @@
 <references/>
-.↑ 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]
-.↑ 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]
-.↑ 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]