title: Neutralizing mutations of carboxylates that bind metal 2 in T5 flap endonuclease result in an enzyme that still requires two metal ions.
abstract: Flap endonucleases (FENs) are divalent metal ion-dependent phosphodiesterases. Metallonucleases are often assigned a "two-metal ion mechanism" where both metals contact the scissile phosphate diester. The spacing of the two metal ions observed in T5FEN structures appears to preclude this mechanism. However, the overall reaction catalyzed by wild type (WT) T5FEN requires three Mg(2+) ions, implying that a third ion is needed during catalysis, and so a two-metal ion mechanism remains possible. To investigate the positions of the ions required for chemistry, a mutant T5FEN was studied where metal 2 (M2) ligands are altered to eliminate this binding site. In contrast to WT T5FEN, the overall reaction catalyzed by D201I/D204S required two ions, but over the concentration range of Mg(2+) tested, maximal rate data were fitted to a single binding isotherm. Calcium ions do not support FEN catalysis and inhibit the reactions supported by viable metal cofactors. To establish participation of ions in stabilization of enzyme-substrate complexes, dissociation constants of WT and D201I/D204S-substrate complexes were studied as a function of [Ca(2+)]. At pH 9.3 (maximal rate conditions), Ca(2+) substantially stabilized both complexes. Inhibition of viable cofactor supported reactions of WT, and D201I/D204S T5FENs was biphasic with respect to Ca(2+) and ultimately dependent on 1/[Ca(2+)](2). By varying the concentration of viable metal cofactor, Ca(2+) ions were shown to inhibit competitively displacing two catalytic ions. Combined analyses imply that M2 is not involved in chemical catalysis but plays a role in substrate binding, and thus a two-metal ion mechanism is plausible.
authors: Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield S3 7HF, United Kingdom.
authors: Christopher G
authors: CG
authors: Tomlinson
authors: Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield S3 7HF, United Kingdom.
authors: Karl
authors: K
authors: Syson
authors: Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield S3 7HF, United Kingdom.
authors: Blanka
authors: B
authors: Sengerová
authors: Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield S3 7HF, United Kingdom.
authors: John M
authors: JM
authors: Atack
authors: Henry Wellcome Laboratories for Medical Research, University of Sheffield School of Medicine and Biomedical Science, Beech Hill Road, Sheffield S10 2RX, United Kingdom.
authors: Jon R
authors: JR
authors: Sayers
authors: Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield S3 7HF, United Kingdom.
authors: Linda
authors: L
authors: Swanson
authors: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720; Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037.
authors: John A
authors: JA
authors: Tainer
authors: Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield S3 7HF, United Kingdom.
authors: Nicholas H
authors: NH
authors: Williams
authors: Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield S3 7HF, United Kingdom. Electronic address: j.a.grasby@sheffield.ac.uk.
authors: Jane A
authors: JA
authors: Grasby
mesh_terms: D001665:Binding Sites; D001703:Biophysics / Q000379:methods; D002118:Calcium / Q000737:chemistry; D002384:Catalysis; D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry; D004798:Enzymes / Q000737:chemistry; D005090:Exodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics*; D007477:Ions; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry*; D008670:Metals / Q000737:chemistry*; D008968:Molecular Conformation; D009154:Mutation; D010710:Phosphates / Q000737:chemistry; D011485:Protein Binding; D012984:Software
chemical_list: D004798:Enzymes; D007477:Ions; D008670:Metals; D010710:Phosphates; D004247:DNA; D005090:Exodeoxyribonucleases; C079044:T5 D15 exonuclease; D008274:Magnesium; D002118:Calcium