Green coffee bean α-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 β-galactosidase. The hydration of d-galactal by the α-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 α-d-galactosides. These results provide an unequivocal assignment of the orientation of an acidic catalytic group to the α-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 β-galactosidase and from that reported for α-glucosidases. Further findings show that d-galacto-octenitol is hydrated by the coffee bean α-ga lactosidase to form the α-anomer of 1,2-dideoxy-d-ga-lactooctulose and by Escherichia coli β-galactosidase to form the β-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.
ASJC Scopus subject areas
- Molecular Biology