Fluoride Inhibition of Enolase: Crystal Structure and Thermodynamics^{<a href="#FN1" rid="FN1" class=" fn">↕</a>}^{<a href="#FN2" rid="FN2" class=" fn">†</a>}

Abstract

It has long been known that fluoride ions inhibit alcoholic fermentation and glycolysis. Warburg and Christian have shown that this is due to the inhibition of enolase (1). Enolase (2-phospho-D-glycerate hydrolyase, EC.4.2.1.11) is an enzyme functioning in the Embden-Meyerhof-Parnas glycolytic pathway that catalyzes the reversible dehydration of 2-phospho-D-glycerate (PGA¹) to yield PEP¹. The enzyme molecule is composed of two identical subunits (2) and has a requirement for two divalent cations per active site for catalytic activity. The first one is often referred to as the “conformational” ion because its binding induces a conformational change. The second ion binds only in the presence of a substrate (or its analogues) and is necessary for enzymatic activity. We refer to it as the “catalytic” ion. These cations are Mg ^{under physiological conditions but can be replaced by a variety of metal ions (}3).

The enolase subunit has been found to exist in three major conformational states. The closed conformation has been observed in the catalytic complex with PGA (4, 5) and with strong inhibitors such as phosphonoacetohydroxamate (6). The semi-closed conformation has been found in the catalytic complex with PEP (4); it differs from the closed conformation in the position of the loop containing His 157, which does not form a direct hydrogen bond with the phosphate moiety but interacts via a water molecule. The open conformation is observed in the absence of substrates or their inhibitory analogues (7).

In addition to its significance for the brewing industry, the fluoride inhibition of enolase has practical ramifications in the treatment of dental plaque in which deep layers are highly anaerobic and dependent on glycolysis for energy requirements and, even more importantly, on the presence of PEP for the transport of sugar by the PEP-dependent phospho-transferase system (8).

From the very beginning, it was suggested that the inhibition of enolase is due to the formation of a magnesium•fluoride•phosphate complex (1). Later, it was found that rabbit muscle enolase was only weakly affected by F ^{alone and that in the presence of P}_i the inhibition was strong and competitive with respect to the substrate (9). These findings were confirmed by studies (10) that showed cooperativity of P_i and F ^{binding and, more specifically, that F is coordinated to the enzyme bound Mg.}

A more detailed study of fluoride binding by yeast enolase was carried out with a fluoride ion-specific electrode by Bunick and Kashket (11). They presented evidence for a binding mechanism in which one F ^{ion was bound per subunit containing conformational Mg ion and P}_i. An additional F ^{could bind only after additional, presumably catalytic, Mg ion bound. The reported dissociation constants for these fluoride ions were 5.0 × 10 and 8.2 × 10, respectively. Addition of the substrate, PGA, led to the release of both bound F ions in a competitive fashion. However, subsequent studies showed that in the presence of PGA, yeast enolase is inhibited by F, though in a complicated, time-dependent fashion (}12), becoming non-competitive over time (cf. reference 10).

There is a large variation in the strength of F ^{inhibition in the presence of different activating cations (}10, 13). The Mg ^{activated yeast enzyme is most strongly inhibited; the Mn activated enzyme is inhibited 40 times more weakly; and the Zn activated enzyme shows no inhibition by F. Mn ion is, however, paramagnetic and enabled utilization of techniques that are not applicable to Mg complexes (}13, 14). These studies quantitatively demonstrated strong positive cooperativity in F ^{and P}_i binding with a 10^{-fold increase in K}_d for F ^{dissociation from the ternary complex yeast enolase-Mn-F and a 10-fold increase in K}_d for P_i dissociation from the enolase•Mn^•P_i complex when compared to the quaternary complex. The data also indicated that one F ^{binds in the active site displacing a water molecule from the first coordination sphere of the bound Mn ion. To explain the potency of the inhibition, Nowak and Maurer suggested that the quaternary complex formation induces a transition state-like conformation of the enzyme (}14).

Zhang et al. reported crystallographic studies of the yeast enolase•fluoride•phosphate complex (15). However, these studies were carried out at a high ammonium sulfate (ca 2 M) concentration, which prohibited binding of the catalytic magnesium ion and presumably the second fluoride ion. Here we report a higher resolution study of the structure of human neuron specific enolase in the presence of fluoride, phosphate and magnesium ions at lower (ca 0.4 M) ionic strength.

Most of the previous studies were carried out using yeast enolase 1 while here investigations of hNSE are reported to take advantage of the superior scattering power of its crystals. It is likely however, that the results are applicable also to the yeast enzyme and vice versa since enolase, like all glycolytic enzymes, is strongly conserved. Yeast enolase 1 and NSE are 62% identical, (73% similar) with two small (two and one residue) deletions present in all known mammalian enolases. All active site residues are conserved (16) and the kinetic parameters are similar (J. M. Brewer and R. Kendrick, unpublished observations).

Inspection of the active site revealed a number of features that are in excellent agreement with previously published solution binding studies (1, 9-14). The structures reported here likely represent the inhibitory complexes formed at physiological conditions. The fluoride/phosphate binding is confirmed by our isothermal titration calorimetry (ITC) study, which is to our knowledge the first thermodynamic documentation of cooperativity between enolase subunits.

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