The roles of Glu186 and Glu380 in the catalytic reaction of soybean beta-amylase.
It has previously been suggested that the glutamic acid residues Glu186 and Glu380 of soybean beta-amylase play critical roles as a general acid and a general base catalyst, respectively. In order to confirm the roles of Glu186 and Glu380, each residue was mutated to a glutamine residue and the crystal structures of the substrate (E186Q/maltopentaose) and product (E380Q/maltose) complexes were determined at resolutions of 1.6 Angstrom and 1.9 Angstrom, respectively. Both mutant enzymes exhibited 16,000- and 37,000-fold decreased activity relative to that of the wild-type enzyme. The crystal structure of the E186Q/maltopentaose complex revealed an unambiguous five-glucose unit at subsites -2 to +3. Two maltose molecules bind on subsites -2 to -1 and +2 to +3 in the E380Q/maltose complex, whereas they bind in tandem to -2 to -1 and +1 to +2 in the wild-type/maltose complex. The conformation of the glucose residue at subsite -1 was identified as a stable (4)C(1) alpha-anomer in the E380Q/maltose complex, whereas a distorted ring conformation was observed in the wild-type/maltose complex. The side-chain movement of Gln380 to the position of a putative attacking water molecule seen in the wild-type enzyme caused the inactivation of the E380Q mutant and an altered binding pattern of maltose molecules. These results confirm the critical roles played by Glu186 in the donation of a proton to the glycosidic oxygen of the substrate, and by Glu380 in the activation of an attacking water molecule. The observed difference between the backbones of E186Q/maltopentaose and E380Q/maltose in terms of Thr342 suggests that the side-chain of Thr342 may stabilize the deprotonated form of Glu186 after the cleavage of the glycosidic bond.
Kang
You-Na
YN
Laboratory of Food Quality Design and Development, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
Adachi
Motoyasu
M
Utsumi
Shigeru
S
Mikami
Bunzo
B
D001665:Binding Sites; D002236:Carbohydrate Conformation; D018360:Crystallography, X-Ray; D018698:Glutamic Acid / Q000378:metabolism*; D006860:Hydrogen Bonding; D008024:Ligands; D008320:Maltose / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D013025:Glycine max / Q000201:enzymology*; D014867:Water / Q000737:chemistry; D001614:beta-Amylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D008024:Ligands; D009844:Oligosaccharides; D010940:Plant Proteins; D014867:Water; C019193:maltopentaose; D018698:Glutamic Acid; D008320:Maltose; D001614:beta-Amylase