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. | <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/ | 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. | ||
<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_211/6residus/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. | <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. | ||
<scene name='Sand_box_211/Lys196/ | <scene name='Sand_box_211/Lys196/3'>Lys196</scene> is positioned between two metal sites. Its mutation perturbs metal ion binding. | ||
<scene name='Sand_box_211/Lys215arg216lys241/ | <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/Lys215216/2'>Lys215 and Arg216</scene> form part of a helix–loop–helix feature. <scene name='Sand_box_211/Arg33/ | <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). | [[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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Following divalent metal ions permit the reaction to take place : <scene name='Sand_box_211/Mn2/1'>Mn2+</scene>, Mg2+, Co2+, <scene name='Sand_box_211/Zn/1'>Zn2+</scene>, Fe2+ and Cu2+. However, the reaction is the most efficient with Mn2+ and Mg2+ as cofactors. Furthermore, it has been shown that T5 5'-exonuclease is able to cleave double-stranded closed-circular plasmids with an Mn2+ cofactor although this enzyme normally is only able to cleave single-stranded 5' ends. | Following divalent metal ions permit the reaction to take place : <scene name='Sand_box_211/Mn2/1'>Mn2+</scene>, Mg2+, Co2+, <scene name='Sand_box_211/Zn/1'>Zn2+</scene>, Fe2+ and Cu2+. However, the reaction is the most efficient with Mn2+ and Mg2+ as cofactors. Furthermore, it has been shown that T5 5'-exonuclease is able to cleave double-stranded closed-circular plasmids with an Mn2+ cofactor although this enzyme normally is only able to cleave single-stranded 5' ends. | ||
The two binding sites for metal ions are located near <scene name='Sand_box_211/8_residus/ | The two binding sites for metal ions are located near <scene name='Sand_box_211/8_residus/4'>acidic residues</scene> (Asp26, Asp68, Glu128, Asp130, Asp153, Asp155, Asp201 and Asp204) which are responsible for binding them. | ||
However, previous studies have shown that the enzyme needs at least three metal ions for the reaction. As most of the T5 5'-exonuclease x-ray structures in the absence of substrate show only two divalent metal ions bound, it implies that the third metal ion binds only in the presence of substrate, to stabilize the enzyme-DNA complex, and has less affinity for the free enzyme. However, the reaction also takes place, if there are only two metal ions present which confirms the two-metal-ion mechanism (figure) and that only two metal ions are needed for the catalytic reaction. | However, previous studies have shown that the enzyme needs at least three metal ions for the reaction. As most of the T5 5'-exonuclease x-ray structures in the absence of substrate show only two divalent metal ions bound, it implies that the third metal ion binds only in the presence of substrate, to stabilize the enzyme-DNA complex, and has less affinity for the free enzyme. However, the reaction also takes place, if there are only two metal ions present which confirms the two-metal-ion mechanism (figure) and that only two metal ions are needed for the catalytic reaction. | ||
The activity of the enzyme changes with the metal ion, which means that for example the T5 5’-exonuclease is more active with Mn2+ than Co2+ or Mg2+ because Mn2+ ions bind most strongly to the protein. | The activity of the enzyme changes with the metal ion, which means that for example the T5 5’-exonuclease is more active with Mn2+ than Co2+ or Mg2+ because Mn2+ ions bind most strongly to the protein. | ||