Purification, characterization and inhibition by fluoride of enolase from Streptococcus mutans DSM 320523.
Enolase from Streptococcus mutans has been purified to homogeneity by a three-step procedure. As shown by analytical ultracentrifugation and polyacrylamide gel electrophoresis under denaturing conditions, the purified enzyme is an octamer with molecular weight Mr = 360 kDa, composed of eight identical subunits with Mr = 45 kDa. The kinetic parameters of S. mutans enolase in the presence of 1 mM Mg2+ are Aspec = 130 IU x mg-1 and KM = 0.44 mM as determined by steady-state experiments in the D-glycerate 2-phosphate to enolpyruvate phosphate reaction. Enzymatic activity is inhibited noncompetitively by fluoride in the range between 0 and 10 mM NaF yielding Ki = Kii = 1.39 mM, but inhibition characteristics become competitive when tested above 10 mM. In the presence of only small amounts of phosphate (0.5 mM) the inhibitory effect of fluoride is enhanced dramatically yielding Ki = 0.26 mM. Inhibition by NaPO3F is competitive with Ki = 0.9 mM, indicating that free fluoride ions in combination with phosphate are more effective in inhibiting S. mutans enolase. It can be concluded that inhibition of enolase by fluoride in combination with phosphate can influence glycolysis in S. mutans and so reduce acid production or even growth rate, thereby leading to potential anticariogenic effects.
Kaufmann
M
M
Universität Witten/Herdecke, BRD.
Bartholmes
P
P
D002021:Buffers; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005459:Fluorides / Q000494:pharmacology*; D010710:Phosphates / Q000494:pharmacology; D010751:Phosphopyruvate Hydratase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D012967:Sodium Dodecyl Sulfate; D012969:Sodium Fluoride / Q000494:pharmacology; D013295:Streptococcus mutans / Q000145:classification / Q000201:enzymology*
D002021:Buffers; D010710:Phosphates; C012980:fluorophosphate; D012967:Sodium Dodecyl Sulfate; D012969:Sodium Fluoride; D010751:Phosphopyruvate Hydratase; D005459:Fluorides