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 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 Glu186, therefore, is clearly indispensable for the liberation of beta-maltose from starch. Substitutions of Leu383 by Ile and Gln, in contrast, led to remarkable increases in the Km 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 kcat/Km of soybean beta-amylase 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 beta-amylase involving the interactions of three essential amino acid residues (Asp101, Glu186 and Glu380) in concert with Leu383, and assumed an indispensable role for Asp101.
Totsuka
A
A
Genetic Engineering Laboratory, Ministry of Agriculture, Forestry and Fisheries, Ibaraki, Japan.
Fukazawa
C
C
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D003505:Cyclodextrins / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013025:Glycine max / Q000201:enzymology* / Q000235:genetics; D047391:alpha-Cyclodextrins; D001614:beta-Amylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D003505:Cyclodextrins; D047391:alpha-Cyclodextrins; D018698:Glutamic Acid; D001614:beta-Amylase; D007930:Leucine; C032613:alpha-cyclodextrin