title: Effects of mono- and divalent metal ions on DNA binding and catalysis of human apurinic/apyrimidinic endonuclease 1.
abstract: Here, we used stopped-flow fluorescence techniques to conduct a comparative kinetic analysis of the conformational transitions in human apurinic/apyrimidinic endonuclease 1 (APE1) and in DNA containing an abasic site in the course of their interaction. Effects of monovalent (K(+)) and divalent (Mg(2+), Mn(2+), Ca(2+), Zn(2+), Cu(2+), and Ni(2+)) metal ions on DNA binding and catalytic stages were studied. It was shown that the first step of substrate binding (corresponding to formation of a primary enzyme-substrate complex) does not depend on the concentration (0.05-5.0 mM) or the nature of divalent metal ions. In contrast, the initial DNA binding efficiency significantly decreased at a high concentration (5-250 mM) of monovalent K(+) ions, indicating the involvement of electrostatic interactions in this stage. It was also shown that Cu(2+) ions abrogated the DNA binding ability of APE1, possibly, due to a strong interaction with DNA bases and the sugar-phosphate backbone. In the case of Ca(2+) ions, the catalytic activity of APE1 was lost completely with retention of binding potential. Thus, the enzymatic activity of APE1 is increased in the order Zn(2+) < Ni(2+) < Mn(2+) < Mg(2+). Circular dichroism spectra and calculation of the contact area between APE1 and DNA reveal that Mg(2+) ions stabilize the protein structure and the enzyme-substrate complex.
authors.affiliation: Institute of Chemical Biology and Fundamental Medicine (ICBFM), Siberian Branch of Russian Academy of Sciences, 8 Lavrentyev Ave., Novosibirsk 630090, Russia. nikita.kuznetsov@niboch.nsc.ru fedorova@niboch.nsc.ru.
authors.forename: Anastasia D
authors.initials: AD
authors.lastname: Miroshnikova
authors.forename: Alexandra A
authors.initials: AA
authors.lastname: Kuznetsova
authors.forename: Yuri N
authors.initials: YN
authors.lastname: Vorobjev
authors.forename: Nikita A
authors.initials: NA
authors.lastname: Kuznetsov
authors.forename: Olga S
authors.initials: OS
authors.lastname: Fedorova
mesh_terms: D001059:Apoproteins / Q000378:metabolism; D055162:Biocatalysis / Q000187:drug effects*; D002413:Cations, Divalent / Q000494:pharmacology*; D002414:Cations, Monovalent / Q000494:pharmacology*; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008958:Models, Molecular; D011188:Potassium / Q000494:pharmacology; D013379:Substrate Specificity / Q000187:drug effects
chemical_list: D001059:Apoproteins; D002413:Cations, Divalent; D002414:Cations, Monovalent; D004247:DNA; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D008274:Magnesium; D011188:Potassium