Sand box 211: Difference between revisions
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<Structure load='1EXN' size='500' frame='true' align='right' caption='T5 5'exonuclease' scene='Insert optional scene name here' /> | <Structure load='1EXN' size='500' frame='true' align='right' caption='T5 5'exonuclease' scene='Insert optional scene name here' /> | ||
T5 5'-exonuclease is a | T5 5'-exonuclease is a homodimeric protein composed of two identical chains,<scene name='Sand_box_211/Vghjvjh/2'>chain a</scene> and <scene name='Sand_box_211/Vhj/1'>chain b</scene>. | ||
Both chains contain a hole, bound by a helical arch composed of two helices in which hydrophobic and positively charged residues are located. The helical arch is situated in front of the active site and only single-stranded DNA can pass through it. Since the enzyme is able to cleave double-stranded DNA, the enzyme has a conformational flexibility to facilitate DNA threading which is required to process the 5' nuclease substrates in the active site. | |||
The core regions of the enzyme are composed of beta-sheets and alpha-helices . These form the base of the active site pocket which bind the catalytic metal ions. | |||
The active site possesses 8 conserved acidic residues (Asp26, Asp68, Glu128, Asp130, Asp153, Asp155, Asp201, Asp204) which interact with divalent metal ions. Tyr82 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 acidic residues (Asp26, Asp68, Glu128, Asp130, Asp153, Asp155, Asp201, Asp204) which interact with divalent metal ions. Tyr82 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. | ||
Six residues (Arg33, Lys83, Arg172, Lys 196, Lys215, Arg216 and Lys241) near the active site permit binding to branched DNA. | Six residues (Arg33, Lys83, Arg172, Lys 196, Lys215, Arg216 and Lys241) near the active site permit binding to branched DNA. | ||
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Lys196 is positioned between two metal sites. Its mutation perturbs metal ion binding. Lys215, Arg216 and Lys241 are important for binding to the 5' overhanging hairpin substrate. Furthermore, residues Lys215 and Arg216 form part of a helix–loop–helix feature. Arg33 binds to a phosphodiester residue in the 3' end of the cleavage site. | Lys196 is positioned between two metal sites. Its mutation perturbs metal ion binding. Lys215, Arg216 and Lys241 are important for binding to the 5' overhanging hairpin substrate. Furthermore, residues Lys215 and Arg216 form part of a helix–loop–helix feature. Arg33 binds to a phosphodiester residue in the 3' end of the cleavage site. | ||
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. They | 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. | ||
Following divalent metal ions permit the reaction to take place : Mn2+, Mg2+, Co2+, Zn2+, 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 : Mn2+, Mg2+, Co2+, Zn2+, 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 acidic residues ( Asp26, Asp68, Glu128, Asp130, Asp153, Asp155, Asp201 and Asp204) which are responsible for binding them. | The two binding sites for metal ions are located near acidic residues ( 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 T5 5' exonuclease is inhibited in presence of Ca2+ ions | 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 T5 5' exonuclease is inhibited in presence of Ca2+ ions. It is a competitive inhibition which means that the Ca2+ binds to the metal binding site and doesn't permit the binding of the catalytic divalent metal ion. | |||
== '''Applications''' == | == '''Applications''' == | ||