Non-competitive Inhibition of Bacterial Phosphofructokinase by Xylitol-5-phosphate

Objectives: Xylitol, a sweetener used in sugar-free gum, is a 5-carbon sugar alcohol that is not metabolized by plaque bacteria. Xylitol enters bacteria via a phosphoenol pyruvate-driven transporter, normally used for fructose, wasting energy to produce xylitol-5-phosphate, which cannot be metabolized and must be expelled. Xylitol or xylitol-5-phosphate also appears to actively depress growth of oral bacteria by inhibiting glycolysis. To investigate this inhibition, we examined the kinetics of the rate-limiting enzyme of glycolysis, phosphofructokinase (PFK), in the presence of xylitol and xylitol-5-phosphate. (In eukaryotic cells, PFK is normally regulated by fructose-2,6-bisphosphate; but in prokaryotic cells by phosphoenol pyruvate.)

Methods: PFK from Bacillus stearothermophilus was purchased from Sigma. The PFK reaction (ATP + fructose-6-phosphate → ADP + fructose-1,6-bisphosphate) was coupled to the oxidation of NADH, using aldolase, triose phosphate isomerase, and glycerol phosphate dehydrogenase, measuring the disappearance of NADH at 340 nm with a Cary 300 spectrophotometer. We prepared xylitol-5-phosphate by incubating xylitol and phosphoenol pyruvate with a protein extract from Streptococcus mutans, and purifying xylitol-5-phosphate by cellulose thin layer chromatography. By systematically varying the concentrations of ATP and fructose-6-phosphate, and fitting the resulting PFK enzyme velocities to hyperbolas, we determined Vmax and the Km for ATP and the Km for fructose-6-phosphate, in the presence or absence of xylitol-5-phosphate.

Results: Sorbitol-6-phosphate, ribose-5-phosphate, fructose-2,6-bisphosphate, and xylitol had no effect on PFK activity. Xylitol-5-phosphate (0.5 mM) inhibited PFK by 44%. The inhibition was non-competitive with both ATP and fructose-6-phosphate. (Phosphoenol pyruvate also inhibited, but was pseudo-competitive with fructose-6-phosphate.)

Conclusion: Non-competitive inhibition of PFK by xylitol-5-phosphate may be a mechanism by which xylitol inhibits glycolysis in oral bacteria. Inhibition of glycolysis would slow bacterial growth and acid production, thereby inhibiting caries.

Supported by NIDCR grant DE05494 and by The University of Tennessee Dental Alumni Fund.