Evidence for a single active site on sugar beet .ALPHA.-glucosidase with maltase and glucoamylase activities.

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Abstract
The active site of sugar beet α-glucosidase catalyzing the hydrolyses of maltose and soluble starch was investigated by kinetic methods. On experiments with mixed substrates, maltose and soluble starch, competition between the two substrates was observed. Lineweaver-Burk plots were linear, and the dependence of V and Km values on the fraction of maltose, f= maltose/(maltose + soluble starch) was in good agreement with that theoretically predicted for a single active site mechanism. From the dependence of V and Km values on pH, the ionization constants of the essential ionizable group 1 and 2 of the free enzyme, pKe1 and pKe2, were determined for each substrate: pKe1=3.9, pKe2=635 for maltose; pKe1 =3J, pKe2=6.5 for soluble starch. Both pKe1 and pKe2 shifted to higher pH with a decrease in the dielectric constant of the reaction mixture. The ionization heat of the ionizable group 2 was nearly zero kcal/mol in either maltose or soluble starch as substrate. Tris, erythritol, methyl-α-glucoside and glucono-δ-lactone competitively inhibited both maltase and glucoamylase activities. The inhibition constants were nearly the same for maltase activity as those for glucoamylase activity. From these results it was concluded that the enzymeattacked maltose and soluble starch by a single active site mechanism.

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