Stereochemistry of D-galactal and D-galacto-octenitol hydration by coffee bean alpha-galactosidase: insight into catalytic functioning of the enzyme.
Green coffee bean alpha-galactosidase was found to catalyze the hydration of D-galactal and (Z)-3,7-anhydro-1,2-dideoxy-D-galacto-oct-2-enitol (D-galacto-octenitol), each a known substrate for beta-galactosidase. The hydration of D-galactal by the alpha-galactosidase in D2O yielded 2-deoxy-2(S)-D-[2-2H]galactose; the hydration of D-[2-2H]galacto-octenitol in H2O yielded 1,2-dideoxy-2(R)-D-[2-2H]galactooct-3-ulose. Thus, the enzyme protonated each substrate from beneath the plane of the ring, as assumed for alpha-D-galactosides. These results provide an unequivocal assignment of the orientation of an acidic catalytic group to the alpha-galactosidase reaction center. In addition, they reveal a pattern of glycal/exocyclic enitol/glycoside protonation by the enzyme that differs from the pattern reported for beta-galactosidase and from that reported for alpha-glucosidases. Further findings show that D-galacto-octenitol is hydrated by the coffee bean alpha-galactosidase to form the alpha-anomer of 1,2-dideoxy-D-galactooctulose and by Escherichia coli beta-galactosidase to form the beta-anomer. That each enzyme converts this enolic substrate to a product whose de novo anomeric configuration matches that formed from its D-galactosidic substrates provides new evidence for the role of protein structure in controlling the steric outcome of reactions catalyzed by these and other glycosylases. The findings are discussed in light of the concept that catalysis by glycosidases involves a "plastic" protonation phase and a "conserved" product configuration phase.
Weiser
W
W
Chemisches Laboratorium, Universität Freiburg, Germany.
Lehmann
J
J
Matsui
H
H
Brewer
C F
CF
Hehre
E J
EJ
D002236:Carbohydrate Conformation; D003069:Coffee / Q000201:enzymology*; D005690:Galactose / Q000031:analogs & derivatives* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D012639:Seeds / Q000201:enzymology; D013379:Substrate Specificity; D013402:Sugar Alcohols / Q000378:metabolism*; D000519:alpha-Galactosidase / Q000378:metabolism*
D003069:Coffee; D013402:Sugar Alcohols; C002038:galactal; C072265:3,7-anhydro-1,2-dideoxygalacto-oct-2-enitol; D000519:alpha-Galactosidase; D005690:Galactose