FUNCTIONAL-ANALYSIS OF GLU380 AND LEU383 OF SOYBEAN BETA-AMYLASE - A PROPOSED ACTION MECHANISM

Soybean beta-amylase, comprising a (beta/alpha)(8)-barrel core with a mobile loop, similar to that of triose phosphate isomerase, was mutate d by site-directed mutagenesis at residues Glu380 and Leu383. X-ray cr ystallographic findings suggest that Glu380 is the counterpart of the catalytic site (Glu186) and that Leu383, located near the active-site cavity, forms an inclusion complex with cyclomaltohexaose. Separate su bstitutions of Glu380 by Gln and Asp completely eliminated the activit y without inducing any significant changes in the circular dichroic sp ectra nor in the binding affinity for cyclomaltohexaose. Glu380, in co operation with Glu186, therefore, is clearly indispensable for the lib eration of beta-maltose from starch. Substitutions of Leu383 by Ile an d Gln, in contrast, led to remarkable increases in the K-m values of b oth mutants when compared to that of the non-mutant enzyme. The mutant s also showed marked reductions in their binding affinities to cycloma ltohexaose. Overall, it would appear that the k(cat)/K-m of soybean be ta-amylase increases in proportion to the length of the substrate mole cule, and depends also on the characteristics of the side chain of the residue at position 383. Leu383, therefore, may be important for both substrate penetration and subsequent retention at the active site. Ba sed on the foregoing, we propose an action mechanism of soybean beta-a mylase involving the interactions of three essential amino acid residu es(Asp101, Glu186 and Glu380) in concert with Leu383, and assumed an i ndispensable role for Asp101.