Fluoride inhibition of yeast enolase. 1. Formation of the ligand complexes

Maurer, Peter; Nowak, Thomas

doi:10.1021/bi00527a024

Cited by 28 publications

(20 citation statements)

References 19 publications

(39 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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 All active site residues are conserved (16) and the kinetic parameters are similar (J. M. Brewer and R. Kendrick, unpublished observations).Inspection of the active site revealed a number of features that are in excellent agreement with previously published solution binding studies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)(10)(11)(12)(13)(14). The structures reported here likely represent the inhibitory complexes formed at physiological conditions.…”

supporting

confidence: 78%

Fluoride Inhibition of Enolase: Crystal Structure and Thermodynamics^,

et al. 2005

View full text Add to dashboard Cite

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...

show abstract

supporting

confidence: 78%

Fluoride Inhibition of Enolase: Crystal Structure and Thermodynamics^,

et al. 2005

View full text Add to dashboard Cite

show abstract

“…The PRR titrations demonstrate that the presence of either one of the ligands, F -or P i , greatly increased the affinity of the second ligand for the enzyme-Mn 2+ ternary complex. This binding synergy is similar to that previously reported (28). These titrations result in the formation of the tightly bound E-Mn 2+ -P i -F -quaternary complex.…”

Section: Resultssupporting

confidence: 90%

“…The mutation has no significant effect on the formation of either ternary or quaternary complexes nor on the synergistic ligand effects observed with F -and P i (Figures 5-7; Table 2). The results of the PRR titrations for the wild-type enolase presented here are similar to those obtained by Maurer and Nowak (28).…”

Section: Resultssupporting

confidence: 89%

Role of His159 in Yeast Enolase Catalysis

Vinarov¹,

Nowak²

1999

Biochemistry

Self Cite

View full text Add to dashboard Cite

There are presently several proposed catalytic mechanisms of yeast enolase, all of which have emerged from separate structural investigations of enolase from yeast and lobster muscle. However, the identities of the residues functioning as the general acid/base pair are not yet established unambiguously. In the Mn(2+)-phosphoglycolate complex of lobster muscle enolase, the imidazole group of His157 (His159 in the yeast enolase numbering system) is in van der Waals contact (4.5 A) with the C(2) of the inhibitor [Duquerroy et al. (1995) Biochemistry 34, 12513-12523]. To gain further information about the role played by His159 in the catalytic mechanism of yeast enolase this residue has been mutated to Ala. The gene encoding for the H159A mutation has been constructed and the mutant protein has been expressed in Escherichia coli. The purified mutant protein is folded properly as indicated by near- and far-UV circular dichroism and fluorescence data, and the mutation has no significant effect on the formation of ternary and quaternary enzyme-ligand complexes. In a typical assay, H159A showed 0.01% of wild-type specific activity, which corresponds to a reduction in k(cat) of 4 orders of magnitude. The H159A fails to ionize the C-2 proton of either 2-PGA or phosphoglycolate. These findings are consistent with His159 serving as a potential catalytic base in the enolase reaction. We have suggested that His159 could also serve as a metal ligand at the third, inhibitory, metal binding site. This proposal is consistent with the catalytic mechanism of yeast enolase. Binding of metal ion at site III interferes with His159 reacting as the catalytic base, i.e., abstracting the C(2) proton from 2-PGA. Metal binding studies support the above proposal. Mn(2+) binding at sites I and II for the His159Ala mutant is identical to that of wild type. The binding of Mn(2+) at the third, inhibitory site of H159A is a factor of 3 weaker compared to wild-type enolase. The factor of 3 in binding is reasonable for the contribution to binding strength of a single nondominant ligand in a chelate [Klemba, M., and Regan, L. (1995) Biochemistry 34, 10094-10100. Regan, L. (1993) Annu. Rev. Biophys. Biomol. Struct. 22, 257-281. Cha et al. (1994) J. Biol. Chem. 269, 2687-2694].

show abstract

“…Inhibitory concentrations of F depend very much on concentrations of Mg2+ and of phosphate, and Warburg and Christian (1942) developed a formula for predicting inhibition, which has been refined on the basis of detailed studies of enzyme-metal-fluoride-phosphate complexes (Mauer and Nowak 1981;Lebioda et al 1993).…”

Section: Effects Of Fluoride On Bacterial Enzymesmentioning

confidence: 99%

Antimicrobial actions of fluoride for oral bacteria

Marquis¹

1995

Can. J. Microbiol.

221

154

View full text Add to dashboard Cite

Fluoride is widely used as a highly effective anticaries agent. Although it is felt that its anticaries action is related mainly to effects on mineral phases of teeth and on the process of remineralization, fluoride also has important effects on the bacteria of dental plaque, which are responsible for the acidification of plaque that results in demineralization. The results of recent studies have shown that fluoride can affect bacterial metabolism through a set of actions with fundamentally different mechanisms. It can act directly as an enzyme inhibitor, for example for the glycolytic enzyme enolase, which is inhibited in a quasi-irreversible manner. Direct action seems also to occur in inhibition of heme-based peroxidases with binding of fluoride to heme. The flavin-based peroxidases of many oral bacteria are insensitive to fluoride. Another mode of action involves formation of metal-fluoride complexes, most commonly AlF4-. These complexes are responsible for fluoride inhibition of proton-translocating F-ATPases and are thought to act by mimicking phosphate to form complexes with ADP at reaction centers of the enzymes. However, the actions of fluoride that are most pertinent to reducing the cariogenicity of dental plaque are those related to its weak-acid character. Fluoride acts to enhance membrane permeabilities to protons and compromises the functioning of F-ATPases in exporting protons, thereby inducing cytoplasmic acidification and acid inhibition of glycolytic enzymes. Basically, fluoride acts to reduce the acid tolerance of the bacteria. It is most effective at acid pH values. In the acidic conditions of cariogenic plaque, fluoride at levels as low as 0.1 mM can cause complete arrest of glycolysis by intact cells of Streptococcus mutans. Overall, the anticaries actions of fluoride appear to be complex, involving effects both on bacteria and on mineral phases. The antibacterial actions of fluoride appear themselves to be complex but to be dominated by weak-acid effects.

show abstract

Fluoride inhibition of yeast enolase. 1. Formation of the ligand complexes

Cited by 28 publications

References 19 publications

Fluoride Inhibition of Enolase: Crystal Structure and Thermodynamics^,

Fluoride Inhibition of Enolase: Crystal Structure and Thermodynamics^,

Role of His159 in Yeast Enolase Catalysis

Antimicrobial actions of fluoride for oral bacteria

Contact Info

Product

Resources

About

Fluoride inhibition of yeast enolase. 1. Formation of the ligand complexes

Cited by 28 publications

References 19 publications

Fluoride Inhibition of Enolase: Crystal Structure and Thermodynamics,

Fluoride Inhibition of Enolase: Crystal Structure and Thermodynamics,

Role of His159 in Yeast Enolase Catalysis

Antimicrobial actions of fluoride for oral bacteria

Contact Info

Product

Resources

About

Fluoride Inhibition of Enolase: Crystal Structure and Thermodynamics^,

Fluoride Inhibition of Enolase: Crystal Structure and Thermodynamics^,