pH-Dependence of Soybean β-Amylase-mediated Reactions

Soybean α-amylase mediates three kinds of reactions: (1) the hydrolysis of amylose, etc.; (2) an esterification of a functional carboxylate of G1u186 with 2, 3-epoxypropyl α-D-glucopyranoside (α-EPG); (3) a hydration of maltal. Thus, what the functional groups for each reaction are, and whether the catalytic sites are the same, are interesting questions. For hydration of maltal, we here studied the binding properties and the pH dependence of the rate parameter (pKm, k2). It was found that the binding of two maltal molecules to the enzyme is required for the hydration, and that only one protonated functional group with pK=6.75 is concerned with the catalysis, although two functional groups with pK=3.5 and pK=8.2 for both the hydrolysis of amylose and the esterification of G1u186 with a-EPG. Hehre et al. (J. Biol. Chem., 261, 2147(1986)) have proposed a possible mechanism for the hydration of maltal catalyzed by sweet potato β-amylase and assumed a functional carboxyl group (pK=3.7) for the hydrolysis of amylose as catalytic group. If this assumption is true in the case of soybean B-amylase, the pK of the functional carboxyl group (normally pK=3.5) must be raised to 6.75 as a result of the maltal binding to the enzyme. From inhibition study with 4-O-α-glucopyranosyl (1→4)-1-deoxynojirimycin as strong competitive inhibitor, it was presumed that three reactions are catalyzed at the same place in the active site.