Isolation, characterization, and inhibition kinetics of enolase from Streptococcus rattus FA-1.

One aspect in a broad spectrum of possible mechanisms of cariostatic reactions of fluoride is its interaction with the metabolism of oral bacteria. Information on the mechanisms and kinetics of fluoride inhibition of essential enzymes of the glycolytic pathway of the relevant bacteria is lacking. In this work, the isolation and purification of enolase from Streptococcus rattus and its characterization are described. The enzyme has been isolated in a monomeric (22 kilodaltons) and dimeric (49 kilodaltons) form. The Km for 2-phosphoglycerate is 4.35 mM. Fluoride inhibition kinetics have competitive character, while phosphate in concentrations above 2 mM and in the presence of 0.5 mM fluoride alters the inhibition kinetics from competitive to noncompetitive. Without fluoride, 2 mM phosphate has a slight stimulatory effect on the enzyme. Monofluorophosphate has a noncompetitive inhibiting effect on the enzyme. This finding suggests that the effect of phosphate may be due to an additional binding of fluoride to the enolase, resulting in a conformational change of the enzyme.

Interactions of micromolar concentrations of fluoride with Streptococcus rattus FA-1.

Inhibition of the metabolic activities of bacteria by trace amounts of fluoride is manifested phenomenologically as changes in the pH gradient and/or the electrical potential between the cellular interior and the surrounding medium. These data were obtained from the intracellular/extracellular distribution of radioactivity labelled fluoride (18F), 5,5-dimethyloxazolidine-2,4-dione (14C), and tetraphenylphosphonium chloride (14C). When taken up from acidic media, trace concentrations of fluoride (1-100 microM) reduce the intracellular/extracellular pH gradient and affect the electrical potential across the cell membrane. The chromatographic fractionation of fluoride-charged bacterial homogenates showed that fluoride is attached to many proteins of the cytoplasm, the cell membrane, and to nonproteinaceous components of the cell wall. Lysozyme treatment synergistically affects the vulnerability of the bacteria to micromolar concentrations of fluoride.