Structure and Function of Soybean β-Amylase

Abstract

Soybean β-amylase was mutated by site-directed mutagenesis at residues (His93, Cys95, Asp101, Glu186, Cys208, Cys343, Glu345, Asp348, Glu380 and Leu383) conserved in highly similar regions of the α-amylase family found in plants and bacteria. The substitutions of Asp101, Glu186 and Glu380 completely eliminated the activity without inducing any significant change in the binding affinity for α-cyclodextrin (α-CD). These data and X-ray crystallographic works confirm Glu186 and Glu380 as catalytic residues of the enzyme. On the other hand, substitutions of Leu383 led to remarkable decreases in the k_cat/K_m values, and those mutants also showed marked reductions in their binding affinity to α-CD. Leu383, therefore, may be important for both substrate penetration and subsequent retention at the active site. Based on the foregoing, we propose an action mechanism of soybean β-amylase involving the interaction of three essential amino acid residues (Asp101, Glu186 and Glu380) in concert with leu383.