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Title: Crystallization of porcine pancreatic elastase and a preliminary neutron diffraction experiment

Abstract

To investigate the structural characteristics of a covalent inhibitor bound to porcine pancreatic elastase (PPE), including H atoms and hydration by water, a crystal of porcine pancreatic elastase with its inhibitor was grown to a size of 1.6 mm{sup 3} for neutron diffraction study. The crystal diffracted to 2.3 Å resolution with sufficient quality for further structure determination owing to the similar atomic scattering properties of deuterium and carbon. Porcine pancreatic elastase (PPE) resembles the attractive drug target leukocyte elastase, which has been implicated in a number of inflammatory disorders. In order to investigate the structural characteristics of a covalent inhibitor bound to PPE, including H atoms and the hydration by water, a single crystal of PPE for neutron diffraction study was grown in D{sub 2}O containing 0.2 M sodium sulfate (pD 5.0) using the sitting-drop vapour-diffusion method. The crystal was grown to a size of 1.6 mm{sup 3} by repeated macroseeding. Neutron diffraction data were collected at room temperature using a BIX-3 diffractometer at the JRR-3 research reactor of the Japan Atomic Energy Agency (JAEA). The data set was integrated and scaled to 2.3 Å resolution in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 51.2,more » b = 57.8, c = 75.6 Å.« less

Authors:
 [1];  [2];  [3]; ;  [2];  [1];  [2];  [4]
  1. Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531 (Japan)
  2. Molecular Structural Biology Group, Quantum Beam Science Directorate, Japan Atomic Energy Agency, 2-4 Shirakata-Shirane, Tokai, Ibaraki 319-1195 (Japan)
  3. Lead Discovery Research Laboratories, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585 (Japan)
  4. (Japan)
Publication Date:
OSTI Identifier:
22360301
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section F; Journal Volume: 63; Journal Issue: Pt 4; Other Information: PMCID: PMC2330211; PMID: 17401204; PUBLISHER-ID: hc5019; OAI: oai:pubmedcentral.nih.gov:2330211; Copyright (c) International Union of Crystallography 2007; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United Kingdom
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; ATOMS; CARBON; CRYSTALLIZATION; DEUTERIUM; DIFFUSION; HYDRATION; MONOCRYSTALS; NEUTRON DIFFRACTION; RESOLUTION; SODIUM SULFATES; SPACE GROUPS

Citation Formats

Kinoshita, Takayoshi, Tamada, Taro, Imai, Keisuke, Kurihara, Kazuo, Ohhara, Takashi, Tada, Toshiji, Kuroki, Ryota, E-mail: kuroki.ryota@jaea.go.jp, and Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531. Crystallization of porcine pancreatic elastase and a preliminary neutron diffraction experiment. United Kingdom: N. p., 2007. Web. doi:10.1107/S1744309107010433.
Kinoshita, Takayoshi, Tamada, Taro, Imai, Keisuke, Kurihara, Kazuo, Ohhara, Takashi, Tada, Toshiji, Kuroki, Ryota, E-mail: kuroki.ryota@jaea.go.jp, & Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531. Crystallization of porcine pancreatic elastase and a preliminary neutron diffraction experiment. United Kingdom. doi:10.1107/S1744309107010433.
Kinoshita, Takayoshi, Tamada, Taro, Imai, Keisuke, Kurihara, Kazuo, Ohhara, Takashi, Tada, Toshiji, Kuroki, Ryota, E-mail: kuroki.ryota@jaea.go.jp, and Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531. Sun . "Crystallization of porcine pancreatic elastase and a preliminary neutron diffraction experiment". United Kingdom. doi:10.1107/S1744309107010433.
@article{osti_22360301,
title = {Crystallization of porcine pancreatic elastase and a preliminary neutron diffraction experiment},
author = {Kinoshita, Takayoshi and Tamada, Taro and Imai, Keisuke and Kurihara, Kazuo and Ohhara, Takashi and Tada, Toshiji and Kuroki, Ryota, E-mail: kuroki.ryota@jaea.go.jp and Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531},
abstractNote = {To investigate the structural characteristics of a covalent inhibitor bound to porcine pancreatic elastase (PPE), including H atoms and hydration by water, a crystal of porcine pancreatic elastase with its inhibitor was grown to a size of 1.6 mm{sup 3} for neutron diffraction study. The crystal diffracted to 2.3 Å resolution with sufficient quality for further structure determination owing to the similar atomic scattering properties of deuterium and carbon. Porcine pancreatic elastase (PPE) resembles the attractive drug target leukocyte elastase, which has been implicated in a number of inflammatory disorders. In order to investigate the structural characteristics of a covalent inhibitor bound to PPE, including H atoms and the hydration by water, a single crystal of PPE for neutron diffraction study was grown in D{sub 2}O containing 0.2 M sodium sulfate (pD 5.0) using the sitting-drop vapour-diffusion method. The crystal was grown to a size of 1.6 mm{sup 3} by repeated macroseeding. Neutron diffraction data were collected at room temperature using a BIX-3 diffractometer at the JRR-3 research reactor of the Japan Atomic Energy Agency (JAEA). The data set was integrated and scaled to 2.3 Å resolution in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 51.2, b = 57.8, c = 75.6 Å.},
doi = {10.1107/S1744309107010433},
journal = {Acta Crystallographica. Section F},
number = Pt 4,
volume = 63,
place = {United Kingdom},
year = {Sun Apr 01 00:00:00 EDT 2007},
month = {Sun Apr 01 00:00:00 EDT 2007}
}
  • The serine protease family of enzymes is one of the most widely studied group of enzymes, as evidenced by the fact that more crystal structures are available for individuals of this superfamily than for any other homologous group of enzymes. These enzymes contain a conserved triad of catalytic residues including Ser-195, His-57, and Asp-102. The active-site serine is very nucleophilic, and serine proteases are inhibited by specific serine protease reagents such as diisopropyl phosphorofluoridate (DFP), phenylmethanesulfonyl fluoride, and 3,4-dichloroisocoumarin. Elastases are a group of proteases that possess the ability to cleave the important connective tissue protein elastin. Elastin has themore » unique property of elastic recoil, is widely distributed in vertebrate tissue, and is particularly abundant in the lungs, arteries, skin, and ligaments. Human neutrophil elastase and pancreatic elastase are two major serine proteases that cleave elastin. Neutrophil elastase is found in the dense granules of polymorphonuclear leukycytes and is essential for phagocytosis and defense against infection by invading microorganisms. Pancreatic elastase is stored as an inactive zymogen in the pancreas and is secreted into the intestines where it becomes activated by trypsin and then participates in digestion. Both elastases cleave substrates at peptide bonds where the P{sub 1} residue is an amino acid residue with a small alkyl side chain.« less
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