X-ray Crystal Structure Analysis of Prolyl Endopeptidase - Inhibitor Complex
| Project/Area Number |
15570095
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Structural biochemistry
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| Research Institution | Tokyo Institute of Technology |
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Principal Investigator |
SATO Takao Tokyo Institute of Technology, Depertment of Life Science, Assistant Professor, 大学院・生命理工学研究科, 助手 (80243731)
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| Project Period (FY) |
2003 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2005: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2004: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2003: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | Prolyl Endopeptidase / Automatic dispenser / Crystal Solubility Curve / Regression Analysis and ANOVA / X-ray Crystal Structure Analysis / Homology Modeling / Docking Simulation / Molecular Orbital Method / プロリルエンドペプチダーゼ / ドッキング・シミュレーション / 沈殿曲線 / 分子軌道計算法 / ストリーク・シーディング / 分子置換法 / 阻害剤-酵素複合体 |
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| Research Abstract |
The Proly Endopeptidase (PEPase) from Flavobacterium meningosepticum is one of the serine protease that cleaves peptide bonds at the carboxyl side of proline residue within which peptides are up to approximately 30 amino acids long. Crystals of PEPase were obtained at 15℃ by vapor diffusion in sitting drops. The protein drops were equilibrated against reservoir solutions containing 22%PEG4000 and buffered at pH7.5 by 0.1M Hepes. The sitting drops contained equal volumes of 8mg/mL enzyme and reservoir solutions. The crystals belong to space group P4_1 and the unit cell dimensions are a=b= 3.17Å, c=169.02Å. There are two molecules in the asymmetric unit. X-ray intensity data to 3.2 Åresolutions were collected at 100K on Rigaku R-Axis IV and were processed with the CrystalClear. The starting model for determination and refinement was that of the porcine brain (PDB code 1H2W). The structure was determined by molecular replacement method with the program AMORE/CCP4 and then refined with the program CNS. Solvent molecules were added once the final crystallographic R-factor and Rfree was lower than 26% and 32%, respectively. The overall fold is the same as that of the asymmetric two molecules, with rms deviations between α-carbon atom positions of 0.04Å. The monomer has a cylindrical shape of an approximate hight of 70Å and diameter of 50 Å, consists of a peptidase domain with its catalytic triads Ser536/Asp620/His655. The domain exhibits a characteristic α/β hydrolase fold and contains a central eight β-strands and is beside the six α-helices. Ser536 is found at the tip of a turn between strand and helix. Such an arrangement referred to as nucleophile elbow. Consequently, the OH group of Ser536 is well accessible to the catalytic imidazole group and nucleophile attacks the aldehyde carbone atom. Asp620 is in the plane of the imidazole ring of His655 which is located in the middle loop.
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Report
(4 results)
Research Products
(28 results)
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[Journal Article] Structural Basis of the Substrate Subsite and the Highly Thermal Stability of Xylanase 10B From Thermotoga maritima MSB82005
Author(s)
Ihsanawati, Kumasaka, T., Kaneko, T., Morokuma, C., Yatsunami, R., Sato, T., et al.
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Journal Title
PROTEINS : Structure, Function, and Bioinformatics 61
Pages: 999-1009
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[Journal Article] A Norovirus Protease Structure Provides Insights into Active and Substrate Binding Site Integrity2005
Author(s)
Nakamura, K., Someya, Y., Kumasaka, T., Ueno, G., Yamamoto, M., Sato, T., et al.
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Journal Title
Journal of Virology 79・21
Pages: 13685-13693
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