Crystalline (monomeric) soybean and (tetrameric) sweet potato β-amylase were shown to catalyze the cis hydration of maltal (α-D-glucopyranosyl-2-deoxy-D-arabino-hex-1-enitol) to form β-2-deoxymaltose. As reported earlier with the sweet potato enzyme, maltal hydration in D2O by soybean β-amylase was found to exhibit an unusually large solvent deuterium kinetic isotope effect (VH/ VD = 6.5), a reaction rate linearly dependent on the mole fraction of deuterium, and 2-deoxy-[2(a)-2H] maltose as product. These results indicate (for each β-amylase) that protonation is the rate-limiting step in a reaction involving a nearly symmetric one-proton transition state and that maltal is specifically protonated from above the double bond. This is a different stereochemistry than reported for starch hydrolysis. With the hydration catalyzed in H2O and analyzed by gas-liquid chromatography, both sweet potato and soybean β-amylase were found to convert maltal to the β-anomer of 2-deoxymaltose. That maltal undergoes cis hydration provides evidence in support of a general-acid-catalyzed, carbonium ion mediated reaction. Of fundamental significance is that β-amylase protonates maltal from a direction opposite that assumed for protonating starch, yet creates products of the same anomeric configuration from both. Such stereochemical dichotomy argues for the overriding role of protein structures in dictating the steric outcome of reactions catalyzed by a glycosylase, by limiting the approach and orientation of water or other acceptors to the reaction center.
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