1999 Fiscal Year Final Research Report Summary
Elucidation of the Catalytic Function of Phospholipases
| Project/Area Number |
09672264
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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 |
Biological pharmacy
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| Research Institution | Osaka University of Pharmaceutical Sciences |
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Principal Investigator |
IKEDA Kiyoshi Osaka University of Pharmaceutical Sciences, Professor, 薬学部, 教授 (50001053)
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| Co-Investigator(Kenkyū-buntansha) |
FUJII Shinobu Osaka University of Pharmaceutical Sciences, research associate, 薬学部, 助手 (80218966)
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| Project Period (FY) |
1997 – 1999
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| Keywords | Phospholipase AィイD22ィエD2 / Sphingomyelinase / Enzyme Inhibitor / Substrate Analog / Catalytic Function |
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| Research Abstract |
1. X-ray crystal structures of bovine pancreas prophospholipase AィイD22ィエD2 (PLAィイD22ィエD2) inhibited by amidetype substrate analogs were determined. The amide group of the inhibitor, which is lacking in the genuine substrate, a strong hydrogen bond was formed between the NH of the inhibitor and the unprotonated NィイD1δ1ィエD1 atom of His 48, which is a catalytic residue of the enzyme.
2. The pH dependence curve of the chemical reaction rate of BPB, which is a potent inhibitor of phospholipase AィイD22ィエD2, with bovine pancreatic PLAィイD22ィエD2 was found to be biphasic. The amino acid residues participating in the two transitions were ascribed to His 48 and the N-terminal α-amino group.
3. We synthesized 3-methoxycarbony-2,4,6-trienal which showed powerful inhibition to PLAィイD22ィエD2. This compound was found by the MALDI-TOF-MS peptide mapping analysis to modify selectively Lys 56 which is included in the interfacial recognition sits of the enzyme.
4. Sphingomyelinase (SMase) from Bacillus cereus was found to have at least two binding sites for MgィイD12+ィエD1 and MgィイD12+ィエD1 binding to the lower affinity site was essential for the catalysis. On the basis of the pH dependence data of the kinetic parameters and the three-dimensional structure of Dnase I, which has a primary structure similar to that of SMase, we proposed a catalytic mechanism for SMase based on general-base catalysis of His 296. Roles of Asp 126 and Asp 156 in the enzyme function of SMase were also studied by using the mutant enzymes. It was demonstrated that deprotonation of Asp 126 enhances the substrate binding and suppresses the catalytic activity, and Asp 156 acts as to decrease the substrate binding and activity.
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Research Products
(18 results)
