Functional Analysis of Glu380 and Leu383 of Soybean β‐Amylase

Abstract

Soybean P-amylase, comprising a @/a),-barrel core with a mobile loop, similar to that of triose phosphate isomerase, was mutated by site-directed mutagenesis at residues Glu380 and Leu383. X-ray crystallographic 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 substitutions of Glu380 by Gln and Asp completely eliminated the activity without inducing any significant changes in the circular dichroic spectra nor in the binding affinity for cyclomaltohexaose. Glu380, in cooperation with Glul86, therefore, is clearly indispensable for the liberation of p-maltose from starch. Substitutions of Leu383 by Ile and Gln, in contrast, led to remarkable increases in the K, values of both mutants when compared to that of the non-mutant enzyme. The mutants also showed marked reductions in their binding affinities to cyclomaltohexaose. Overall, it would appear that the k,,JK,,, of soybean pamylase increases in proportion to the length of the substrate molecule, 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. Based on the foregoing, we propose an action mechanism of soybean P-amylase involving the interactions of three essential amino acid residues (AsplOl, Glu186 and Glu380) in concert with Leu383, and assumed an indispensable role for AsplOl.

Crystal Structure of Recombinant Soybean β-Amylase Complexed with β-Cyclodextrin

“…The structural analysis of the soybean maltose-␤-amylase complex indicated that Glu 186 and Glu 380 play important roles in the enzymatic reaction as general acid and base catalysts, respectively (16). This finding is supported by the results of site-directed mutagenesis (17,18) and affinity labeling (19). In addition, the structures of ␣-CD⅐␤-amylase and maltose⅐␤-amylase complexes revealed that a flexible loop plays a key role in the reaction.…”

“…Leu 383 , which forms an inclusion complex, is one of the residues that contribute to the slipping mechanism (12). Totsuka et al (18) suggested by site-directed mutagenesis that Leu 383 may work to bind polymeric substrate as a winder (18). Probably, Leu 383 plays an important role in the single chain attack mechanism as well as in the binding of polymeric substrate, because the residue seems to block the cleaved substrate from release into the solvent (see Fig.…”

Structural and Enzymatic Analysis of Soybean β-Amylase Mutants with Increased pH Optimum

“…The structural analysis of the soybean ␤-amylase (SBA) 1 -maltose complex indicated that Glu 186 and Glu 380 play important roles in the enzymatic reaction as a general acid and a base catalyst, respectively (16). This finding is supported by the results of site-directed mutagenesis (22) and affinity labeling (23). In the case of B. cereus ␤-amylase (BCB), Glu 172 and Glu 367 act as the general acid and base catalyst, respectively, corresponding to Glu 186 and Glu 380 in soybean ␤-amylase (20,21).…”

Purification, characterization, immunolocalization and structural analysis of the abundant cytoplasmic β‐amylase from Calystegia sepium (hedge bindweed) rhizomes

“…The three‐dimensional model of the C. sepium β‐amylase strongly resembles that of the soybean [33,39] and sweet potato [38]β‐amylases and shares the typical (α/β) 8 barrel core which is common to all other β‐amylases of different origins [44]. According to both structural [33,39] and functional data [45–47], four amino‐acid residues (Asp101, Glu186, Glu345 and Glu380) located in a cleft occurring between the (α/β) 8 barrel core and the smaller globular region play a key role in the catalytic activity of the soybean β‐amylase. These four residues are conserved in all other plant β‐amylases including the C. sepium β‐amylase.…”

“…origins [44]. According to both structural [33,39] and functional data [45][46][47], four amino-acid residues (Asp101, Glu186, Glu345 and Glu380) located in a cleft occurring between the (a/b) 8 barrel core and the smaller globular region play a key role in the catalytic activity of the soybean b-amylase. These four residues are conserved in all other plant b-amylases including the C. sepium b-amylase.…”