Study on the Enzyme Action Mechanism in the Phospholipid Hydrolysis by Means of small Molecular Inhibitors

小分子抑制剂水解磷脂的酶作用机制研究

• 批准号: 09480145
• 负责人: KATSUMURA Shigeo
• 金额: $ 3.46万
• 依托单位: Kwansei Gakuin University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 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-09480145/
• 关键词: phospholipase AィイD22ィエD2; sphingomyelinase; synthesis of enzyme inhibitors; elucidation of inhibitory mechanism; interfacial recognition site; oxazolidinone substrate analog; synthesis of sphingomyelin; sphingolipid analog inhibitor; スフィンゴミエリナーゼ阻害

Phospholipids are classified into glycerophospholipids and sphingophospholipids. Phospholipase AィイD22ィエD2 (PLAィイD22ィエD2) catalyzes the hydrolysis of the ester linkage at the sn-2 position of glycerophospholipids and responsibles for the metabolism of unsaturated fatty acids. Meanwhile, sphingomyelinase is the general term of the enzyme which catalyzes the hydrolysis of the phosphoester linkage of sphingomyelin to produce ceramide, which has been known as a lipid second messenger in mammalian cell membranes.We found that 3-methoxycarbonyl-2, 4, 6-trienal (A) strongly inactivated phospholipase AィイD22ィエD2 which was isolated from bovine pancreas. The inactivation mechanism of PLAィイD22ィエD2 by the compound A was elucidated by means of the stereocontrolled synthesis of the derivatives of A, structure activity relationship, amino acid analysis of the complex with PLAィイD22ィエD2, and model reactions with amines. Thus, the compound A was found to selectively modify Lys-56 which is included in the … More interfacial recognition site of bovine pancreatic PLAィイD22ィエD2, by the MALDI-TOF-Mass peptide mapping analyses of the complex.We also synthesized the new cyclic amide analog as the second-generation inhibitor having an oxazolidinone ring, because we had previously synthesized the oxazolidinone phospholipid analog which competitively acted on the catalytic site of PLAィイD22ィエD2.On the other hand, in the study of sphingomyelinase, we achieved the efficient synthesis of (D)-erythro-sphigomyelin, and found that (D)-erythro stereochemistry is essentially important as the substrate for sphingomyelinase and the double bond in the backbone skeleton was not essential for the hydrolysis by B. cereus sphingomyelinase. Furthermore, we established the stereocontrolled efficient synthesis of sphingomyelin methylene and ethylene analogs, which are the first substrate-analog inhibitors, and they actually inhibited the hydrolytic ability of the enzyme. These analog inhibitors might be useful for the elucidation of the sphingomyelinase hydrolytic mechanism. Less

磷脂分为甘油磷脂和鞘磷脂。磷脂酶A （PLA）可催化甘油磷脂sn-2位酯链的水解，并负责不饱和脂肪酸的代谢。同时，鞘磷脂酶是催化鞘磷脂磷酸链水解生成神经酰胺的酶的总称，神经酰胺被认为是哺乳动物细胞膜中的脂质第二信使。我们发现3-methoxycarbonyl-2 4 6-trienal (A)强烈灭活磷脂酶ィイD22摊位ィエD2从牛胰腺分离。通过A衍生物的立体控制合成、结构活性关系、与PLA配合物的氨基酸分析以及与胺的模拟反应，阐明了化合物A对PLA的失活机理。因此,这种化合物被发现有选择地修改Lys-56包含在 ... 更多界面识别网站的牛胰腺解放军ィイD22摊位ィエD2,由MALDI-TOF-Mass肽图分析的复杂。我们还合成了新的环酰胺类似物作为第二代具有恶唑烷酮环的抑制剂，因为我们之前已经合成了恶唑烷酮磷脂类似物，该类似物竞争性地作用于PLA的催化位点。另一方面，在鞘磷脂酶的研究中，我们实现了(D)- red -sphigomyelin的高效合成，发现(D)- red - thro立体化学作为鞘磷脂酶的底物至关重要，骨干骨架中的双键对于蜡状芽孢杆菌鞘磷脂酶的水解并不是必需的。此外，我们还建立了立体控制高效合成鞘磷脂亚甲基和乙烯类似物的方法，这是第一个底物类似物抑制剂，它们实际上抑制了酶的水解能力。这些类似抑制剂可能有助于阐明鞘磷脂酶的水解机制。少

项目成果

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Murakami，M.等：“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”Bioorg。

Fujii,S.;Tani,T.:Inoue,S.;Iwama,S.;Katsumura,S.;Ikeda,K.: "pH Dependence of the Reaction Rate of p-Bromophenacyl Bromide and of the Binding Constant of Ca^<2+> and an Amide-Type Substrate Analog to Bovine Panacreatic Phospholopase A_2," Arch.Biochem.Bioph

Fujii,S.;Tani,T.:Inoue,S.;Iwama,S.;Katsumura,S.;Ikeda,K.：“对溴苯甲酰溴反应速率和 Ca^ 结合常数的 pH 依赖性

Fujii, S. ; Tani, T. ; Inoue, S. ; Iwama, S. ; Katsumura, S. : Ikeda, K.: "pH Dependence of the Reaction Rate of p-Bromophenacyl Bromide and the Binding Constant of Ca^<2+> and an Amide-Type Substrate Analog to Bovine Pancreatic Phospholipase A_2"Arch. Bi

藤井，S.；

Tanaka, K.; Kamatani, M.; Mori, H.; Fujii, S.; Ikeda, K.; katsumura, S.: "The Inhibitory Mechanism of Bovine Pancreatic Phospholipase AィイD22ィエD2 by Aldehyde Terpenoids."Tetrahedron. 55. 1657-1686 (1999)

Tanaka，K.；Kamatani，H.；Fujii，S.；katsumura，S.：“醛类萜的抑制机制”。 1686 (1999)

Tomoo,K.: "X-Ray Crystal Structure Determination and Molecular Dynamics Simulation of Phospholipase A_2 Inhibited by Amide-type Substrate Analogues." Biochimimica et Biophysica Acta. 1340. 178-186 (1997)

Tomoo,K.：“酰胺型底物类似物抑制磷脂酶 A_2 的 X 射线晶体结构测定和分子动力学模拟。”