A. Totsuka et C. Fukazawa, FUNCTIONAL-ANALYSIS OF GLU380 AND LEU383 OF SOYBEAN BETA-AMYLASE - A PROPOSED ACTION MECHANISM, European journal of biochemistry, 240(3), 1996, pp. 655-659
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.