Crystal Structure of Catalytic Site Mutant of beta-Amylase from Bacillus cereus var. mycoides Cocrystallized with Maltopentaose

摘要

The X-ray crystal structure of a catalytic site mutant of beta-amylase, E172A (Glu172->Ala), from Bacillus cereus var. mycoides complexes with a substrate, maltopentaose (G5), and the wild-type enzyme complexes with maltose were determined at 2.1 and 2.0 A resolution, respectively. Clear and continuous density corresponding to G5 was observed in the active site of E172A, and thus, the substrate, G5, was not hydrolyzed. All glucose residues adopted a relaxed ~4C_1 conformation, and the conformation of the maltose unit for Glc2 and Glc3 was much different from those of other maltose units, where each glucose residue of G5 is named Glc1-Glc5 (Glc1 is at the nonreducing end). A water molecule was observed 3.3 A from the C1 atom of Glc2, and 3.0 A apart from the OE1 atom of Glu367 which acts as a general base. In the wild-type enzyme-maltose complex, two maltose molecules bind at subsites -2 and -1 and at subsites +1 and +2 in tandem. The conformation of the maltose molecules was similar to that of the condensation product of soybean beta-aymlase, but differed from that of G5 in E172A. When the substrate flips between Glc2 and Glc3, the conformation energy of the maltose unit was calculated to be 20 kcal/mol higher than that of the cis conformation by MM3. We suggest that beta-amylase destabilizes the bond that is to be broken in the ES complex, decreasing the activation energy, DELTAG~= not, which is the different in free energy between this state and the transition state.