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Elucidation of the Catalytic Function of Phospholipase A_2 and C

磷脂酶 A_2 和 C 催化功能的阐明

基本信息

批准号：
07672399
负责人：
IKEDA Kiyoshi
金额：
$ 1.6万
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：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

1. Effects of Ca^<2+> on the bindings of Group I and II PLA_2s to a genuine substrate, an amide-type substrate analog, or an oxazolidinone-type analog were studied. The Ca^<2+> dependency in the binding of the gunuine substrate to the both types of PLA_2s were found to be very similar to that for the oxazolidinone-type substrate analog, but differed greatly from that for the amide-type substrate analog. 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 bovine pancreatic PLA_2 by a manoalide (MLD) -analog were investigated. It was found that Lys-56 of the enzyme was modified and this modification was responsible for the enzyme inactivation.3. pH dependences of the kinetic parameters for the hydrolysis of Lyso-PC,catalyzed by PC-PLC,were studied. One and three transitions were observed on the pH dependence curves of 1/K_m and k_<cat>, respectively. Although similar results were obtained also by using PC as a substrate, the extents pK sifts of an ionizable group accompanying the bindings of Lyso-PC and PC were different from each other.4. pH dependences of the kinetic parameters for the hydrolysis of HNP,catalyzed by Bacillus cereus sphingomyelinase (SMase) and their mutants (D126G and D156G), were studied. The results suggested that the Asp-126 participated in the substrate binding and catalysis, and the Asp-156 reduced the binding ability of HNP and catalytic efficiency.

1.研究了Ca^2+对Ⅰ型和Ⅱ型PLA_2与真正底物、酰胺型底物类似物或恶唑烷酮类类似物结合的影响。在与两种PLA_2结合时，Gunuine底物的Ca^2+依赖性与恶唑烷酮类底物类似物非常相似，但与酰胺类底物类似物有很大差异。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.研究了一种新的磷脂酰肌醇（MLD）类似物对牛胰磷脂酶A_2（PLA_2）的化学修饰及失活作用。发现酶的赖氨酸-56被修饰，这种修饰是导致酶失活的原因.研究了PC-PLC催化Lyso-PC水解动力学参数的pH依赖性。在1/K_m和K_（1/K_m）的pH依赖曲线上分别观察到一个和三个转变<cat>。虽然使用PC作为底物也获得了类似的结果，但Lyso-PC和PC结合时伴随的可电离基团的pK值彼此不同。研究了蜡状芽孢杆菌鞘磷脂酶（SMase）及其突变体（D126 G和D156 G）催化水解HNP的动力学参数与pH的关系。结果表明，Asp-126参与了HNP与底物的结合和催化作用，而Asp-156降低了HNP与底物的结合能力和催化效率。