Three metal ions participate in the reaction catalyzed by T5 flap endonuclease

Abstract

Protein nucleases and RNA enzymes depend on divalent metal ions to catalyze the rapid hydrolysis of phosphate diester linkages of nucleic acids during DNA replication, DNA repair, RNA processing, and RNA degradation. These enzymes are widely proposed to catalyze phosphate diester hydrolysis using a "two-metal-ion mechanism." Yet, analyses of flap endonuclease (FEN) family members, which occur in all domains of life and act in DNA replication and repair, exemplify controversies regarding the classical two-metal-ion mechanism for phosphate diester hydrolysis. Whereas substrate-free structures of FENs identify two active site metal ions, their typical separation of > 4 A appears incompatible with this mechanism. To clarify the roles played by FEN metal ions, we report here a detailed evaluation of the magnesium ion response of T5FEN. Kinetic investigations reveal that overall the T5FEN-catalyzed reaction requires at least three magnesium ions, implying that an additional metal ion is bound. The presence of at least two ions bound with differing affinity is required to catalyze phosphate diester hydrolysis. Analysis of the inhibition of reactions by calcium ions is consistent with a requirement for two viable cofactors (Mg2+ or Mn2+). The apparent substrate association constant is maximized by binding two magnesium ions. This may reflect a metal-dependent unpairing of duplex substrate required to position the scissile phosphate in contact with metal ion(s). The combined results suggest that T5FEN primarily uses a two-metal-ion mechanism for chemical catalysis, but that its overall metallobiochemistry is more complex and requires three ions.

FIGURE 1.

The two-metal-ion mechanism proposed for reactions of metallonucleases and the active site of T5FEN showing the varying positions of metal ions in FEN structures. a, two metal-ion mechanism. One metal ion acts as a source of nucleophilic hydroxide ion and binds to a non-bridging oxygen of the scissile phosphate diester acting as an electrophilic catalyst. A second metal ion is coordinated to the leaving group oxygen assisting with leaving group departure and binds to the same non-bridging oxygen of the scissile phosphate diester. b, active site structure of T5FEN (1UT5, gray with purple carboxylate residues) illustrating the loop of the helix-three-turn-helix (H3TH) (teal) motif and seven active site carboxylates present in similar positions in all FENs and the eighth carboxylate (D201) present in the active sites of bacteriophage and bacterial enzymes (see supplemental Fig. S1). Two metal ions, M1 and M2 (purple) are bound with a separation of 8 Å. Metal ions observed in structures of T4FEN (1TFR, cyan), MjFEN (Methanococcus jannaschii, 1A77, rose), hFEN (1UL1x, green), positioned by overlay, are shown. Although all FENs conserve seven active site carboxylates, the position of M2 observed in each structure is variable.

FIGURE 2.

Variation of the steady state catalytic parameters of T5FEN as a function of magnesium ion concentration. Experiments were conducted at pH 9.3 and 37 °C with varying amounts of MgCl₂ and KCl to maintain constant ionic strength. All measurements represent the result of three independent experiments with standard errors shown. a, variation in k_cat as a function of magnesium ion concentration. Slopes of 0 (red), 1 (blue), and 2 (green) are illustrated. The data have been fit to Equation 2 (“Results”), yielding K_DMgES1 = 0.04 ± 0.003 mm and K_DMgES2 = 2.3 ± 0.06 mm and (k_cat)_max = 146 ± 1 min^-1. b, variation in 1/K_m as a function of magnesium ion concentration fitted to Equation 3 with K_DMgE1 = K_DMgE2 (black curve, yields K_DMgE1 = K_DMgE2 0.22 ± 0.02 mm and (1/K_m)_max = 0.03 ± 0.003 nm^-1) and Equation 4 where n = 2 (red curve, yields = 0.07 ± 0.007 mm², and (1/K_m)_max = 0.03 ± 0.003 nm^-1). c, variation in k_cat/K_m for as a function of magnesium ion concentration fitted to Equation 5 with = 0.07 mm² (black curve, yields K_DMgE2 = 4.9 ± 1.4 mm and (k_cat/K_m)_max = 4.2 ± 1 min^-1 nm^-1) and Equation 6 with K_DMgE1 = K_DMgE2 = 0.21 mm (blue curve, yields K_DMgE3 = 6.0 ± 1.8 mm and (k_cat/K_m)_max = 4.6 ± 1.3 min ^-1 nm^-1). Data shown as black circles are derived from the individual catalytic parameters whereas data shown as red circles are the observed rate constant at low concentrations of substrate where 29[S] < [Mg]/10.

FIGURE 3.

The inhibitory effects of added calcium ions on Mg²⁺- and Mn²⁺-supported T5FEN reactions. Experiments were conducted at 37 °C with varying amounts of CaCl₂ and KCl to maintain the same ionic strength in all experiments. Reactions supported by Mg²⁺ were studied at pH 9.3, whereas those supported by Mn²⁺ were studied at pH 7.5 as described under “Experimental Procedures.” a, variation in k_obs/k₀ as a function of calcium ion concentration where k_obs is the observed normalized initial rate (v/[E]) at a given concentration of Ca²⁺ and k₀ is v/[E] in the absence of Ca²⁺. Reactions contained 0.1 mm Mg²⁺ (triangles), 2 mm Mg²⁺ (diamonds), or 0.1 mm Mn²⁺ (circles). Data were fitted to Equation 7 to yield respective values of apparent K_I (the concentration of Ca²⁺ where k_obs/k₀ = 0.5) of 0.037 ± 0.002 mm, 0.22 ± 0.02 mm, and 1.2 ± 0.1 mm. b, the variation in k_obs/k₀ as a function of Ca²⁺ concentration for a reaction containing 0.1 mm Mn²⁺. Combined standard errors for k_obs/k₀ are shown. The black line shows the best fit to a simple competitive inhibition scheme where one Ca²⁺ displaces one Mn²⁺ (Equation 7). The red line is a slope of -2 indicating a dependence on 1/[Ca²⁺]² at high Ca²⁺. c, variation in k_obs/k₀ as a function of Ca²⁺ concentration for a reaction containing 2 mm Mg²⁺. Combined standard errors for k_obs/k₀ are shown. The black line shows the best fit to Equation 7. The red line is a slope of -2 indicating a dependence on 1/[Ca²⁺]² at high Ca²⁺.