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.
Highlights
Flap endonucleases (FENs)4, essential in all life forms, are members of the 5Ј-nuclease superfamily of structure-specific
If two metal ions that participate in chemical catalysis are coordinated to the central carboxylate of the T5FEN active site (Asp-130), positioned as those observed in hFEN1 structures (Fig. 1) (2, 4), two-metal ion catalysis mechanisms where both metals coordinate the scissile phosphate diester could take place
The work described here supports the hypothesis that the major rate acceleration in the reactions catalyzed by flap endonucleases requires the presence of two metal ions
Summary
Materials—Wild type (WT) and D201I/D204S T5FENs and 5Ј-overhanging hairpin substrate (5Ј-FAM-pd(CGCTGTCGAACACACGCTTGCGTGTGTTC)) (HP5F) were prepared and purified to homogeneity as described (3, 24, 25). Plots of v/[E] versus [S], where [S] Ͻ Km, were used to determine kcat/Km. Calcium Inhibition—For magnesium-supported T5FEN reactions, reaction mixtures containing 0.5 or 2 mM MgCl2, 1 M HP5F, 25 mM potassium glycinate, pH 9.3, 0.1 mg/ml BSA, varying [CaCl2] with the appropriate amount of KCl to keep ionic strength constant as above, were initiated by the appropriate amounts of T5FEN (0.3–30 nM). Mixtures containing 0.5 or 2 mM MgCl2, 0.5 M HP5F, 25 mM CHES, pH 9.3, 0.1 mg/ml BSA, CaCl2 with the appropriate amount of KCl were initiated by the addition of the appropriate amount of D201I/D204S T5FEN (final concentration 5– 40 nM). Experiments contained 2 mM EDTA or varying concentrations of CaCl2 (0.5–10 mM), with the appropriate amount of KCl as for kinetic analyses, 100 nM HP5F (for WT T5FEN) or 10 nM HP5F (for D201I/D204S), 25 mM HEPES, pH 7.5, or 25 mM potassium glycinate, pH 9.3, 0.1 mg/ml BSA, and 1 mM DTT.
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