Fluoride inhibition of enolase: crystal structure and thermodynamics

Abstract

Enolase is a dimeric metal-activated metalloenzyme which uses two magnesium ions per subunit: the strongly bound conformational ion and the catalytic ion that binds to the enzyme-substrate complex inducing catalysis. The crystal structure of the human neuronal enolase-Mg2F2P(i) complex (enolase fluoride/phosphate inhibitory complex, EFPIC) determined at 1.36 A resolution shows that the combination of anions effectively mimics an intermediate state in catalysis. The phosphate ion binds in the same site as the phosphate group of the substrate/product, 2-phospho-D-glycerate/phosphoenolpyruvate, and induces binding of the catalytic Mg2+ ion. One fluoride ion bridges the structural and catalytic magnesium ions while the other interacts with the structural magnesium ion and the ammonio groups of Lys 342 and Lys 393. These fluoride ion positions correspond closely to the positions of the oxygen atoms of the substrate's carboxylate moiety. To relate structural changes resulting from fluoride, phosphate, and magnesium ions binding to those that are induced by phosphate and magnesium ions alone, we also determined the structure of the human neuronal enolase-Mg2P(i) complex (enolase phosphate inhibitory complex, EPIC) at 1.92 A resolution. It shows the closed conformation in one subunit and a mixture of open and semiclosed conformations in the other. The EPFIC dimer is essentially symmetric while the EPIC dimer is asymmetric. Isothermal titration calorimetry data confirmed binding of four fluoride ions per dimer and yielded Kb values of 7.5 x 10(5) +/- 1.3 x 10(5), 1.2 x 10(5) +/- 0.2 x 10(5), 8.6 x 10(4) +/- 1.6 x 10(4), and 1.6 x 10(4) +/- 0.7 x 10(4) M(-1). The different binding constants indicate negative cooperativity between the subunits; the asymmetry of EPIC supports such an interpretation.

Figure 1

Composite omit map (2F_oF_c) of EFPIC structure at 2.5 σ level.

Figure 2

Omit electron density map (F_oF_c) in the active site contoured at a 5.0 σ level. The Mg ions and water molecules, fluoride ions and the phosphate ion coordinated to them were omitted from the model.

Figure 3

Interatomic distances in the active site. The values for subunits A and B (in parentheses) are given.

Figure 4

Superposition (based on the enzyme C_α atoms) of the yeast enolase•PEP complex (PDP entry 2ONE) on the active site of EFPIC. The proposed positions of fluoride ions (in pale green) overlap with the oxygen atoms of the carboxylate of PEP (in pale blue). Also, there is an excellent correspondence between the positions of the inhibitory phosphate ion (in purple) and the phosphate moiety of PEP. The magnesium ions, Mg1 conformational and Mg2 catalytic, are shown in silver and water molecules in red.

Figure 5

Superposition of the EFPIC in cyan (subunit A) and orange (subunit B) and the “native” (hNSE•2Mg²⁺•P_i/hNSE•Mg²⁺•Cl⁻) complex in blue (subunit A) and yellow (subunit B). The ligands are shown only for the EFPIC structure using the same colors as in Figure 4. It is apparent that subunits A are very similar but large differences between catalytic loops are present in subunits B.

Figure 6

Isothermal titration of hNSE with fluoride. A. The upper panel shows raw data. The lower panel shows the enthalpogram retrieved from these data; the line represents the least-squares fit to a four sequential binding sites model with a Χ² value of 3.4 × 10³. B. Fit to a two sets of two identical sequential binding sites yielded a Χ² value of 2.6 × 10⁵. C. Fit to two independent binding sites, each with a multiplicity of 2, yielded a Χ² value of 5.6 × 10⁵.

Figure 6

Isothermal titration of hNSE with fluoride. A. The upper panel shows raw data. The lower panel shows the enthalpogram retrieved from these data; the line represents the least-squares fit to a four sequential binding sites model with a Χ² value of 3.4 × 10³. B. Fit to a two sets of two identical sequential binding sites yielded a Χ² value of 2.6 × 10⁵. C. Fit to two independent binding sites, each with a multiplicity of 2, yielded a Χ² value of 5.6 × 10⁵.