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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...

Like most cellular RNA enzymes, the bI5 group I intron requires binding by a protein cofactor to fold correctly. Here, we use single-molecule approaches to monitor the structural dynamics of the bI5 RNA in real time as it assembles with its CBP2 protein cofactor. These experiments show that CBP2 binds to the target RNA in two distinct modes with apparently opposite effects: a "non-specific" mode that forms rapidly and induces large conformational fluctuations in the RNA, and a "specific" mode that forms slowly and stabilizes the native RNA structure. The bI5 RNA folds though multiple pathways toward the native state, typically traversing dynamic intermediate states induced by non-specific binding of CBP2. These results suggest that the protein cofactor-assisted RNA folding involves sequential non-specific and specific protein-RNA interactions. The non-specific interaction potentially increases the local concentration of CBP2 and the number of conformational states accessible to the RNA, which may promote the formation of specific RNA-protein interactions.

In the presence of magnesium, enolase catalyzes the dehydration of 2-phospho-D-glycerate (PGA) to phosphoenolpyruvate (PEP) in glycolysis and the reverse reaction in gluconeogensis at comparable rates. The structure of human neuron specific enolase (hNSE) crystals soaked in PGA showed that the enzyme is active in the crystals and produced PEP; conversely soaking in PEP produced PGA. Moreover, the hNSE dimer contains PGA bound in one subunit and PEP or a mixture of PEP and PGA in the other. Crystals soaked in a mixture of competitive inhibitors tartronate semialdehyde phosphate (TSP) and lactic acid phosphate (LAP) showed asymmetry with TSP binding in the same site as PGA and LAP in the PEP site. Kinetic studies showed that the inhibition of NSE by mixtures of TSP and LAP is stronger than predicted for independently acting inhibitors. This indicates that in some cases inhibition of homodimeric enzymes by mixtures of inhibitors (“heteroinhibition”) may offer advantages over single inhibitors.