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.测定了酰胺型底物类似物抑制牛胰腺磷脂酶A原D22酶D2（PLA酶D22酶D2）的X-射线晶体结构。抑制剂的酰胺基在真正的底物中是缺乏的，抑制剂的NH与His 48的未质子化的N-氨基-D1δ1-氨基-D1原子之间形成强氢键，His 48是酶的催化残基. BPB是磷脂酶A β D22 β D2的有效抑制剂，其与牛胰腺PLA β D22 β D2的化学反应速率的pH依赖性曲线被发现是双相的。参与这两个转变的氨基酸残基归属于His 48和N-末端α-氨基.合成了3-甲氧羰基-2，4，6-三烯醛，它对聚乳酸（PLA）过氧化物酶D_（22）过氧化物酶D_（22）有较强的抑制作用。通过MALDI-TOF-MS肽图分析发现该化合物选择性修饰了酶的界面识别位点Lys 56.蜡状芽孢杆菌（Bacillus cereus）鞘磷脂酶（SMase）至少有两个Mg ~（2+）D_1结合位点，Mg ~（2+）D_1与低亲和力位点的结合是该酶催化的关键。根据动力学参数的pH依赖性数据和具有与SMase类似的一级结构的Dnase I的三维结构，我们提出了基于His 296的一般碱催化的SMase催化机理。利用突变体酶研究了Asp 126和Asp 156在SMase酶功能中的作用。结果表明，Asp 126的去质子化增强了底物结合，抑制了催化活性，Asp 156的作用是降低底物结合和活性。

项目成果

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。”

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。”

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。”

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。

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”J。