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Article

Fluoride Inhibition of Enolase: Crystal Structure and Thermodynamics^†^,^‡

Jie Qin,^§ Geqing Chai,^§ John M. Brewer, Leslie L. Lovelace,^§ and Lukasz Lebioda*^§

Department of Chemistry and Biochemistry and Center for Colon Cancer Research, University of South Carolina, Columbia, South Carolina 29208, and Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602

Biochemistry, 2006, 45 (3), pp 793–800

DOI: 10.1021/bi051558s

Publication Date (Web): December 22, 2005

†

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. Department of Energy, Office of Basic Energy Sciences, under Contract W-31-109-Eng-38.

‡

Atomic coordinates have been deposited with the Protein Data Bank as entries 2AKZ and 2AKM for the structure of the fluoride/phosphate complex and the phosphate complex, respectively.

Department of Chemistry and Biochemistry, University of South Carolina.

Department of Biochemistry and Molecular Biology, University of Georgia.

Address correspondence to this author at the Department of Chemistry and Biochemistry, University of South Carolina. Phone: (803) 777-2140. Fax: (803) 777-9521. E-mail: lebioda@ mail.chem.sc.edu.

Center for Colon Cancer Research, University of South Carolina.

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 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 Å 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 K_b values of 7.5 × 10⁵ ± 1.3 × 10⁵, 1.2 × 10⁵ ± 0.2 × 10⁵, 8.6 × 10⁴ ± 1.6 × 10⁴, and 1.6 × 10⁴ ± 0.7 × 10⁴ M^-¹. The different binding constants indicate negative cooperativity between the subunits; the asymmetry of EPIC supports such an interpretation.