Crystalline Aspergillus niger α-glucosidase and highly purified preparations of rice α-glucosidase II and Trichoderma reesei trehalase were found to catalyze the hydration of [2-2H]-D-gluco-octenitol, i.e., (Z)-3,7-anhydro-1,2-dideoxy-[2-2H]-D-gluco-oct-2-enitol, to yield 1,2-dideoxy-[2-2H]-D-gluco-octulose. In each case, the stereochemistry of the reaction was elucidated by examining the newly formed centers of asymmetry at C-2 and C-3 of the hydration product. The C-l to C-3 fragment of each isolated [2-2H] -D-giuco-octulose product was recovered as [2-2H] propionic acid and identified by its positive optical rotatory dispersion as the S isomer, showing that each enzyme had protonated the octenitol (at C-2) from above its re face. 'H NMR spectra of enzyme/D-gluco-octenitol digests in D20 showed that the α-anomer of [2-2H]-D-gluco-octulose was exclusively produced by each α-glucosidase, whereas the β-anomer was formed by action of the trehalase. The trans hydration catalyzed by the α-glucosidases was found to be very strongly inhibited by the substrate; the cis hydration reaction catalyzed by the trehalase showed no such inhibition. Special importance is attached to the finding that in hydrating octenitol each enzyme creates a product of the same anomeric form as in hydrolyzing an α-D-glucosidic substrate. This result adds substantially to the growing evidence that individual glycosylases create the configuration of their reaction products by a means that is independent of donor substrate configuration, that is, by a means other than “retaining” or “inverting” substrate configuration.
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