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 Å 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 catalytic Mg 2+ 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-Mg 2 P i complex (enolase phosphate inhibitory complex, EPIC) at 1.92 Å resolution. It shows the closed conformation in one subunit and a mixture of open and semi-closed conformations in the other. The EPFIC dimer is essentially symmetric while EPIC dimer is asymmetric. Isothermal titration calorimetry data confirmed binding of four fluoride ions per dimer and yielded K b values of 7.5 × 10 5 ± 1.3 × 10 5 , 1.2 × 10 5 ± 0.2 × 10 5 , 8.6 × 10 4 ± 1.6 × 10 4 , 1.6 × 10 4 ± 0.7 × 10 4 M −1 . The different binding constants indicate negative cooperativity between the subunits; the asymmetry of EPIC supports such an interpretation. Keywords enolase; fluoride inhibition; negative cooperativity; glycolysis; crystal structure; isothermal titration calorimetry 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-↕ Atomic coordinates have been deposited with the Protein Data Bank as entries 2AKZ and 2AKM for the structure of fluoride/phosphate complex and the phosphate complex respectively. † This work was supported in part by NIH grant CA076560. Data were collected at the Southeast Regional Collaborative Access Team (SER-CAT) 22-BM beamline at the Advanced Photon Source, Argonne National Laboratory. Supporting institutions may be found at www.ser-cat.org/members.html. Use of the Advanced Photon Source was supported by the U. S. phospho-D-glycerate hydrolyase, EC.4.2.1.11) is an enzyme functioning in the EmbdenMeyerhof-Parnas glycolytic pathway that catalyzes the reversible dehydration of 2-phospho-D-glycerate (PGA 1 ) to yield PEP 1 . 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 b...

Stopped‐flow studies of the reaction of d‐tartronate semialdehyde‐2‐phosphate with human neuronal enolase and yeast enolase 1

“…There is considerable crystallographic evidence [8,[22][23][24]] that binding of substrate/analogues at the active sites of both enzymes is accompanied by movement of polypeptide loops that close around the active sites. (Since crystals are routinely prepared with saturating levels of all ligands, it is not presently possible to say whether substrate binding alone -no catalytic Mg 2+ bound -produces any loop(s) movement, but it is plausible that some movement occurs [7].)…”

Fluoride Exposure May Accelerate the Osteoporotic Change in Postmenopausal Women: Animal Model of Fluoride-induced Osteoporosis

“…The scientific justification for F therapy for osteoporosis, which induced irregular periosteal bone formation, ligament calcification, abnormal mineralization, and an increase of osteoid formation [29][30][31][32][33][34][35][36], is as follows: Concerning the high dose of F treatment, the increase of bone mineral density might be attributed to the pathological mineralization seen by an abnormal calcification and the presence of large crystals located outside the collagen fibrils [16,17,35,37]. This occurs because a high content of F ions absolutely inhibits the synthesis or activity of certain enzymes such as enolase and carbonic anhydrase [13,14,[38][39][40][41][42]. This might be fatal to cellular metabolism and eventually lead to pathological calcification, which might be similar to the vascular calcification accompanying cellular degeneration [43].…”

Comparative study of storage compound breakdown in germinating seeds of three lupine species

“…Such intensive inhibitory effect of NaF on 14 CO 2 liberation (as well as on radioactivity of ethanolsoluble and -insoluble fractions; Tables 2, 3, 4) is probably a result of non-specific action of NaF. Enolase is inhibited by direct binding of fluoride to the enzyme (Qin et al 2006). However, fluoride is considered as an agent that binds Ca ?2 and Mg ?2 , and in that way decreases availability of these ions to many enzymes.…”

“…1), but NaF inhibits glycolysis as well. Both processes are inhibited at the enolase (phosphoenolpyruvate hydratase) stage (Miller 1993;Qin et al 2006). We tested mixture of mitochondrial electron transport chain inhibitors (KCN, NaN 3 and SHAM) known to suppress mitochondrial respiration, and MSO as a glutamine synthetase inhibitor which restricts amino acids synthesis (Masclaux-Daubresse et al 2006) (Fig.…”