NDLI: Neutralizing Mutations of Carboxylates That Bind Metal 2 in T5 Flap Endonuclease Result in an Enzyme That Still Requires Two Metal Ions*

Content Provider	PubMed Central
Author	Tomlinson, Christopher G. Karl, Syson Sengerová, Blanka Atack, John M. Sayers, Jon R. Swanson, Linda Tainer, John A. Williams, Nicholas H. Grasby, Jane A.
Copyright Year	2011
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 Mg2+ 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 Mg2+ 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 [Ca2+]. At pH 9.3 (maximal rate conditions), Ca2+ substantially stabilized both complexes. Inhibition of viable cofactor supported reactions of WT, and D201I/D204S T5FENs was biphasic with respect to Ca2+ and ultimately dependent on 1/[Ca2+]2. By varying the concentration of viable metal cofactor, Ca2+ 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.
Related Links	http://dx.doi.org/10.1074/jbc.M111.230391
Starting Page	30878
File Format	PDF
ISSN	1083351X
e-ISSN	1083351X
Journal	The Journal of Biological Chemistry
Issue Number	35
Volume Number	286
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	2011-09-02
Access Restriction	Open
Rights Holder	American Society for Biochemistry and Molecular Biology
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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