Elucidation of the Catalytic Function of Phospholipase A_2 and C

1. 研究了Ca^<2+>对I和II族PLA_2s与真底物、酰胺型底物类似物或恶唑烷酮型类似物结合的影响。原物底物与两类PLA_2s结合的Ca^<2+>依赖性与恶唑烷酮型底物类似物非常相似，但与酰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。研究了马诺酸（MLD）类似物对牛胰腺PLA_2的化学改性和失活。发现酶的Lys-56发生了修饰，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_<cat>的pH依赖性曲线上分别观察到1次和3次跃迁。虽然用PC作为底物也得到了类似的结果，但Lyso-PC和PC结合的可电离基团的pK筛选程度不同。研究了蜡样芽孢杆菌鞘磷脂酶（SMase）及其突变体D126G和D156G对HNP水解动力学参数的pH依赖性。结果表明，Asp-126参与了底物的结合和催化，Asp-156降低了HNP的结合能力和催化效率。

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.