Kinetic evidence for two active sites in beta-D-fucosidase of Helicella ericetorum.
1. The kinetics of beta-D-fucosidase of the snail H. ericetorum have been studied. The enzyme shows beta-D-fucosidase, beta-D-glucosidase and beta-D-galactosidase activities, all associated in a single peak in both DEAE-cellulose chromatography and isoelectric focusing (p1 4.35), having the same optimal pH (5.0). 2. With the corresponding p-nitrophenyl glycosides as substrates, beta-D-fucosidase activity shows the lowest Km, the highest Vmax and the best Vmax/Km value; close activity values were obtained for beta-D-glucosidase, however, beta-D-galactosidase activity is much lower in this enzyme. 3. All the kinetic evidence suggests that this enzyme has two active sites: a fuco-gluco site and a galacto site. 4. beta-D-fucosidase and beta-D-glucosidase activities have similar Km, Vmax, Vmax/Km and Ki values; these values are very different from those of beta-D-galactosidase activity. beta-D-fucosides and beta-D-glucosides completely compete for a common active site in mixed-substrate experiments, while beta-D-galactosides only partially compete with both glycosides. 5. With delta-D-gluconolactone, the enzyme shows a hyperbolic mixed-type inhibition, mainly competitive for beta-D-fucosidase and beta-D-glucosidase activities (with the same inhibition sub-type), and predominantly non-competitive for beta-D-galactosidase activity (with different inhibition sub-type). With delta-D-gluconolactone more inhibition of beta-D-fucosidase and beta-D-glucosidase activities was found, and with gamma-D-galactonolactone, more inhibition of beta-D-galactosidase activity was detected. 6. The enzyme is activated by some carbohydrates, probably in relation with a transglycosylation mechanism.
D000818:Animals; D001665:Binding Sites; D002241:Carbohydrates / Q000494:pharmacology; D055598:Chemical Phenomena; D002621:Chemistry; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D012908:Snails / Q000201:enzymology*; D005644:alpha-L-Fucosidase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D002241:Carbohydrates; C028219:beta-D-fucosidase; D005644:alpha-L-Fucosidase