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

Abstract

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.

Quasi-irreversible inhibition of enolase of Streptococcus mutans by flouride

“…The data presented show clearly that fluoride can irreversibly inhibit enolase of permeabilized cells of S. mutans GS-5, and this type of inhibition may be related to the non-competitive inhibition reported by others [8,9]. The term 'irreversible' has to be qualified because of the finding that inhibition could be reversed by phosphoenolpyruvate or by very high concentrations of 2-phosphoglycerate.…”

“…Recent studies of the inhibitory effects of fluoride on enolase isolated from mutans streptococci [8,9] have led to the conclusion that inhibition is complex and has both competitive and noncompetitive components. These findings of complex inhibition led us to reinvestigate fluoride inhibition of enolase but with use of permeabilized cells that allow for easy washing of the inhibited enzyme to remove unbound or loosely bound fluoride.…”

Cloning, expression and characterization of an extracellular enolase fromLeuconostoc mesenteroides

“…Fluoride is an inhibitor of L. mesenteroides enolase, with a K i of 5.4, 4.6 and 9.2 mM in 0, 1 and 20 mM phosphate, respectively. The K i values are significantly higher than inhibition constants for enolases from S. rattus (K i $0.85 mM Hüther et al, 1990) and from carp muscle (K i $0.24 mM Pietkiewicz & Kustrzeba-Wójcicka, 1983) in the absence of phosphate. Moreover, low concentrations of phosphate greatly enhance fluoride inhibition of yeast enolase .…”

“…Because enolase is a heat shock protein, its high thermostability is not unusual. Similarly, highly thermostable yeast enolase and Streptococcus rattus (Hüther et al, 1990) enolase have been characterized. Enolases from higher organisms (carp, rabbit muscle and bovine brain), however, are less resistant to thermal denaturation (Wold & Ballou, 1957;Pietkiewicz & Kustrzeba-Wójcicka, 1983;Nazarian et al, 1992;Kustrzeba-Wójcicka & Golczak, 2002).…”

“…Subunit molecular weights of most enolases are c. 45kDa (Wold & Ballou, 1957;Holt & Wold, 1961;Westhead & McLain, 1964;Wold, 1971;Wang & Himoe, 1974;Faller et al, 1977;Pietkiewicz & Kustrzeba-Wójcicka, 1983;Brewer, 1985;Kaufmann & Bartholmes, 1992;Peak et al, 1994;Schurig et al, 1995;Brown et al, 1998a). Enolase from Streptococcus rattus is evidently an exception to the general trend, being a dimer of native mass 49 kDa (Hüther et al, 1990). Prokaryotic a-enolases are highly conserved proteins that may function extracellularly in patho-physiological processes (Pancholi, 2001).…”