1v3i
The roles of Glu186 and Glu380 in the catalytic reaction of soybean beta-amylase
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OverviewOverview
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.
About this StructureAbout this Structure
1V3I is a Single protein structure of sequence from Glycine max with SO4 as ligand. Active as Beta-amylase, with EC number 3.2.1.2 Full crystallographic information is available from OCA.
ReferenceReference
The roles of Glu186 and Glu380 in the catalytic reaction of soybean beta-amylase., Kang YN, Adachi M, Utsumi S, Mikami B, J Mol Biol. 2004 Jun 18;339(5):1129-40. PMID:15178253
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