1994 Fiscal Year Final Research Report Summary
Analysis of beta-Amylase Action by X-Ray Crystallography
| Project/Area Number |
06044121
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| Research Category |
Grant-in-Aid for international Scientific Research
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| Allocation Type | Single-year Grants |
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| Section | Joint Research |
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| Research Institution | Kyoto University |
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Principal Investigator |
MIKAMI Bunzo Kyoto University, Assistant Professor, 食糧科学研究所, 助教授 (40135611)
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| Co-Investigator(Kenkyū-buntansha) |
SACCHETTINI ジェイムズ シイ Albert Einstein College of Medicine, Assistant
HEHRE Edward j. Albert Einstein College Medicine, Professor Emeritus, Professor
SACCHETTINI James c. Albert Einstein College Medicine, Assistant Professor
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| Project Period (FY) |
1994
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| Keywords | beta-amylase / X-ray crystallography / enzyme mechanism / enzyme-substrate complex / フレキシブルループ |
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| Research Abstract |
beta-Amylase is one of the classic enzyme which liberates beta-anomeric maltose from the non-reducing ends of alpha-1,4-glucans such as starch by inverting substrate anomeric configuration. We have first determined the three-dimensional structure of soybean beta-amylase complexed with its competitive inhibitor, alpha-cyclodextrin. In the present study, we have determined the structure of soybean beta-amylase (SBA) complexed with its substrate analogs, maltose (product), maltal (hydration substrate) and glucose in order to elucidate the catalytic mechanism of beta-amylase.
Trigonal SBA crystals were prepared after 600 mg of isozyme 2 was isolated and purified from defatted soybean meal by ammonium sulfate fractionation and ion exchange chromatography. The crystals were soaked to the mother solution containing maltose, maltal or glucose in order to prepare respective complexed forms. Their diffraction data were collected by a area detector at Albert Einstein College of Medicine to less th
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an 2.0* resolution. The structures were refined with the protein moiety of SBA/alpha-cyclodextrin complex as a initial structure. The final models containing 490 amino acid residues, each substrate analogs and about 200-300 water molecules gave R-factors of 0.15-0.17. From the final model of SBA/maltose complex, it was found that two molecules of maltose successively bind to subsites 1-4. Part of the subsites were occupied by maltotetraose which was produced by the reverse reaction. It was clearly identified that two Glu residues, Glu186 and Glu380 near the cleavage site have the role of catalysis. The side chains of Glu186 and Glu380 make hydrogen bonds to O of the cleaving glycosyl linkage and O1 of the product maltose, respectively. It was hypothesized that water molecule which is activated by the side chain of Glu380 attacks to the C1 of glucose moiety at subsite 2. The final model of SBA/maltal complex showed that two molecules of 2-deoxy maltose (hydration product of maltal) bind to the same subsites 1-4, suggesting that the hydrolysis and the hydration occur at the same site. The model of SBA/glucose complex prepared at low concentration of glucose showed only one glucose at subsite 1, but high concentration of glucose made it possible to bind three molecules of glucose at subsite 1,3 and 4, suggesting that substrate conformational change is required to bind to subsite 2. In the final models of maltose, maltal and glucose complexes, a flexible loop (Gly96-Ile103) is found to be "closed" conformation, though this loop was first found to be "open" in the SBA/alpha-cyclodextrin complex. The role of this flexible loop is to fix the transition state of the substrate by making hydrogen bond between the side chain of Asp 101 and O2 of glucose residue at subsite 1 and to release product maltose in the "open" state. The enzyme kinetics in the ammonium sulfate solution also supported the view of the binding of the substrate analogs to the enzyme. Less
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Research Products
(9 results)
