Elucidation of the Catalytic Function of Phospholipases

磷脂酶催化功能的阐明

• 批准号: 09672264
• 负责人: IKEDA Kiyoshi
• 金额: $ 1.98万
• 依托单位: Osaka University of Pharmaceutical Sciences
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09672264/
• 关键词: Phospholipase AィイD22ィエD2; Sphingomyelinase; Enzyme Inhibitor; Substrate Analog; Catalytic Function

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.

1。牛胰腺预防磷脂酶AI D2的X射线晶体结构。 2。发​​现BPB的化学反应是磷脂酶AI D2的电源.3。 Mg II D12+Yi D1和Mg II D12+EIER D1与PHE参数的基础结合和DNase I的三维结构。基于他的296的一般基础催化。ASP126在使用tatant酶研究的Smase Werso的酶功能中的作用。底物结合和活性。

项目成果

Shinobu Fujii: "Catalytic and Toxicity Mechanisms of Secretory Phospholipase A_2." J.Toxicol.-Toxin Reviews. 17(3). 279-313 (1998)

Shinobu Fujii：“分泌型磷脂酶 A_2 的催化和毒性机制。”

Seiji Iwama: "Design and synthesis of new secretory phospholipase A_2 inhibitor of a phospholipid analog." Bioorg.Med.Chem.Lett.8. 3495-3498 (1998)

Seiji Iwama：“磷脂类似物的新型分泌型磷脂酶 A_2 抑制剂的设计和合成。”

Masahiro Murakami: "An efficient synthesis of short-chain sphingomyelin analogs and their susceptibility to hydrolysis catalyzed by sphingomyelinase." Bioorg.Med.Chem.Lett.7(13). 1725-1728 (1997)

Masahiro Murakami：“短链鞘磷脂类似物的有效合成及其对鞘磷脂酶催化水解的敏感性。”

Katsunori Tanaka: "The inhibitory mechanism of bovine pancreatic phospholipase AィイD22ィエD2 by aldehyde terpenoids."Tetrahedron. 55 (6). 1657-1686 (1999)

Katsunori Tanaka：“醛类四面体对牛胰腺磷脂酶 A-D22 的抑制机制。”55 (6) (1999)。

Shinobu Fujii: "pH Dependence of the reaction rate of p-bromophenacyl bromide and of the binding constants of Ca^<2+> and an amide-type substrate analog to bovine pancreatic phospholipase A_2"Arch. Biochem. Biophys.. 354(1). 73-82 (1998)

Shinobu Fujii：“对溴苯甲酰溴的反应速率以及Ca 2 和酰胺型底物类似物与牛胰磷脂酶A_2的结合常数的pH依赖性”Arch。