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Elucidation of the Catalytic Function and the Inhibition mechanism of Phospholipase A_2

磷脂酶A_2的催化功能和抑制机制的阐明

基本信息

批准号：
05671853
负责人：
IKEDA Kiyoshi
金额：
$ 1.28万
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 General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05671853/
关键词：
Phospholipase A_2 Enzyme Inhibitor Substrate Analog Manoalide Catalytic Function Calcium Ion

项目摘要

1. Effects of Ca^<2+> and pH on the kinetic parameter for the hydrolysis of monodispersed 1,2-dihexanoy1-sn-glycero-3-phosphorylcholine, catalyzed by Group I and II phospholipases A_2 (PLA_2s) , were studied by the pH-stat assay method in the absence or presence of carbonic amidetype, oxazolidinone-type, and sulfonic amide-type substrate analogs. The Ca^<2+> dependency and participation of the catalytic group His 48 in the binding of genuine substrate to both types of PLA_2s were found to be very similar to those of the oxazolidinone-type substrate analog, but differed greatly from the carbonic amide-type and sulfonic amide-type substrate analogs. This finding suggests that the binding mode of oxazolidinone-type substrate analog is very similar to that of the genuine substrate.2. Chemical modification and inactivation of Group I and II PLA_2s were investigated by the use of a manoalide (MLD) -analog. It was found that Lys-56 of bovine pancreatic PLA_2s was modified by MLD-analog and that this modification was responsible for enzyme inactivation. It was indicated that the inactivation of pancreatic and N.naja atra PLA_2s originated from the modification of Lys residues at the interfacial recognition site, and that the inactivation of P.australis, T.flavoviridis and V.russelli russelli PLA_2s arose from the modification of Lys residues at the catalytic site, interfacial recognition site and regions outside both sits. The inactivation of A.halys blomhoffii PLA_2s was assumed to be due to the modification of Lys residues outside the two sites described above. We thought that the modification of the Lys residues outside the above two sites give rise to conformational changes leading to inactivation

1.在碳酰胺型、恶唑烷酮型和磺酰胺型底物类似物存在或不存在的情况下，用pH-STAT法研究了Ca~(2+)和pH对单分散磷脂酶A_2(PLA_2S)催化1，2-dihexanoy1-sn-glycero-3-phosphorylcholine，水解动力学参数的影响。催化基团His-48与两种类型的磷脂酶A_2结合的催化基团His-48对Ca~(2+)的依赖性和参与程度与恶唑烷酮型底物类似物非常相似，但与碳酰胺和磺胺型底物类似物有很大不同。这一发现表明，恶唑烷酮类底物类似物的结合模式与真正的底物非常相似。用甘露内酯(MLD)类似物研究了第一组和第二组PLA2的化学修饰和失活。结果发现，牛胰腺磷脂酶A_2s的Lys-56被MLD-类似物修饰，这种修饰是导致酶失活的原因。结果表明，胰腺和中华眼镜蛇PLA2S的失活源于界面识别位点的Lys残基的修饰，而南美白念珠菌、黄绿色葡萄球菌和russelli russelli PLA2的失活源于催化位点、界面识别位点和两个位点外区域的Lys残基的修饰。该酶的失活可能是由于上述两个位点外的赖氨酸残基的修饰所致。我们认为，Lys残基在上述两个位点外的修饰会引起构象变化，导致失活