Crystal Structure of a Catalytic Site Mutant of -Amylase from Bacillus cereus var. mycoides Cocrystalli
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- 作者：Hideo Miyake ; Genji Kurisu ; Masami Kusunoki ; Sigenori Nishimura ; Shinichi Kitamura ; and Yasunori Nitta
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：May 20, 2003
- 年：2003
- 卷：42
- 期：19
- 页码：5574 - 5581
- 全文大小：504K
- 年卷期：v.42,no.19(May 20, 2003)
- ISSN：1520-4995
文摘
The X-ray crystal structure of a catalytic site mutant of 
-amylase, E172A (Glu172 
Ala),from Bacillus cereus var. mycoides complexed with a substrate, maltopentaose (G5), and the wild-typeenzyme complexed with maltose were determined at 2.1 and 2.0 Å resolution, respectively. Clear andcontinuous 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 4C1 conformation, and the conformationof the maltose unit for Glc2 and Glc3 was much different from those of other maltose units, where eachglucose residue of G5 is named Glc1-Glc5 (Glc1 is at the nonreducing end). A water molecule wasobserved 3.3 Å from the C1 atom of Glc2, and 3.0 Å apart from the OE1 atom of Glu367 which acts asa general base. In the wild-type enzyme-maltose complex, two maltose molecules bind at subsites -2and -1 and at subsites +1 and +2 in tandem. The conformation of the maltose molecules was similar tothat of the condensation product of soybean -amylase, but differed from that of G5 in E172A. When thesubstrate flips between Glc2 and Glc3, the conformational energy of the maltose unit was calculated tobe 20 kcal/mol higher than that of the cis conformation by MM3. We suggest that -amylase destabilizesthe bond that is to be broken in the ES complex, decreasing the activation energy, 
G
, which is thedifference in free energy between this state and the transition state.
-amylase, E172A (Glu172 Ala),from Bacillus cereus var. mycoides complexed with a substrate, maltopentaose (G5), and the wild-typeenzyme complexed with maltose were determined at 2.1 and 2.0 Å resolution, respectively. Clear andcontinuous 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 4C1 conformation, and the conformationof the maltose unit for Glc2 and Glc3 was much different from those of other maltose units, where eachglucose residue of G5 is named Glc1-Glc5 (Glc1 is at the nonreducing end). A water molecule wasobserved 3.3 Å from the C1 atom of Glc2, and 3.0 Å apart from the OE1 atom of Glu367 which acts asa general base. In the wild-type enzyme-maltose complex, two maltose molecules bind at subsites -2and -1 and at subsites +1 and +2 in tandem. The conformation of the maltose molecules was similar tothat of the condensation product of soybean -amylase, but differed from that of G5 in E172A. When thesubstrate flips between Glc2 and Glc3, the conformational energy of the maltose unit was calculated tobe 20 kcal/mol higher than that of the cis conformation by MM3. We suggest that -amylase destabilizesthe bond that is to be broken in the ES complex, decreasing the activation energy, G, which is thedifference in free energy between this state and the transition state.