Sand box 211: Difference between revisions
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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 <scene name='Sand_box_211/8_residus/ | The two binding sites for metal ions are located near <scene name='Sand_box_211/8_residus/3'>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. | ||