title: Two divalent metal ions in the active site of a new crystal form of human apurinic/apyrimidinic endonuclease, Ape1: implications for the catalytic mechanism.
abstract: The major human abasic endonuclease, Ape1, is an essential DNA repair enzyme that initiates the removal of apurinic/apyrimidinic sites from DNA, excises 3' replication-blocking moieties, and modulates the DNA binding activity of several transcriptional regulators. We have determined the X-ray structure of the full-length human Ape1 enzyme in two new crystal forms, one at neutral and one at acidic pH. The new structures are generally similar to the previously determined structure of a truncated Ape1 protein, but differ in the conformation of several loop regions and in spans of residues with weak electron density. While only one active-site metal ion is present in the structure determined at low pH, the structure determined from a crystal grown at the pH optimum of Ape1 nuclease activity, pH 7.5, has two metal ions bound 5 A apart in the active site. Enzyme kinetic data indicate that at least two metal-binding sites are functionally important, since Ca(2+) exhibits complex stimulatory and inhibitory effects on the Mg(2+)-dependent catalysis of Ape1, even though Ca(2+) itself does not serve as a cofactor. In conjunction, the structural and kinetic data suggest that Ape1 catalyzes hydrolysis of the DNA backbone through a two metal ion-mediated mechanism.
authors: Molecular and Structural Biology Division, Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, CA, 94550, USA.
authors: P T
authors: PT
authors: Beernink
authors: B W
authors: BW
authors: Segelke
authors: M Z
authors: MZ
authors: Hadi
authors: J P
authors: JP
authors: Erzberger
authors: D M
authors: DM
authors: Wilson
authors: B
authors: B
authors: Rupp
mesh_terms: D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D002413:Cations, Divalent / Q000378:metabolism*; D003067:Coenzymes / Q000378:metabolism; D003460:Crystallization; D018360:Crystallography, X-Ray; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008670:Metals / Q000378:metabolism*; D008958:Models, Molecular; D009038:Motion; D010084:Oxidation-Reduction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
chemical_list: D002413:Cations, Divalent; D003067:Coenzymes; D004268:DNA-Binding Proteins; D008670:Metals; D004247:DNA; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D008274:Magnesium; D002118:Calcium