TRANSPORT OF D-FRUCTOSE AND ITS ANALOGS BY TRYPANOSOMA-BRUCEI

Kinetic parameters for entry of D-fructose into Trypanosoma brucei brucei have been determined. The net uptake of D-fructose was found to be rapid and occurred at a rate which was comparable with that observed for uptake of D-glucose. The K(m) and V(max) were 3.91 +/- 1.58 mM and 69.1 +/- 7.2 nmol min-1 (mg protein)-1. D-Fructose was metabolized to pyruvate under aerobic conditions and to pyruvate and glycerol under anaerobic conditions in a manner similar to D-glucose. Comparisons of the kinetic parameters for D-fructose transport and metabolism indicated that uptake was rate limiting. Inhibition constants (K(i)) for inhibition of 6-deoxy-D-glucose by D-fructose and D-fructose transport by 6-deoxy-D-glucose were consistent with the K(m) values for these two substrates. These interactions indicate that D-fructose and 6-deoxy-D-glucose share a single common transporter. 1,5-Anhydro-D-glucitol and 1,5 anhydro-D-mannitol (the fused pyranose ring analogues of D-glucose and D-mannose) have been found to interact well with the transporter, while L-sorbose (a D-fructose analogue with a pyranose ring) had only low affinity. However, 2,5-anhydro-D-mannitol (a fused furanose ring analogue Of D-fructose) inhibited both 6-deoxy-D-glucose and D-fructose transport with a K(i) of approx.0.8 mM. The high affinity for 2,5-anhydro-D-mannitol (2-deoxy-D-fructofuranose) indicates that D-fructose is transported in the furanose ring form. This was directly demonstrated by measuring inhibition of 6-deoxy-D-glucose transport by beta-D-fructopyranose solutions during the approach to anomeric equilibrium with beta-D-fructofuranose. We therefore propose that the trypanosome transporter accommodates D-glucose as a pyranose ring and D-fructose as a furanose ring by binding to hydrogen bonds directed at the C-3, C-4 and ring oxygen positions and that a bulky group may be tolerated at C-2. The ring oxygen is however not essential, as 2R,5R-dihydroxymethyl-3R4R-dihydroxypyrollidine (a fructofuranose analogue with a ring nitrogen) was also accepted by the trypanosome transporter.