Elucidation of the Catalytic Function of Phospholipase A_2 and C

1.研究了Ca~(2+)~(2+)与真底物、酰胺类底物或恶唑烷酮类类似物结合的影响。谷氨酸底物与两种类型的PLA2结合的钙依赖性与恶唑烷酮型底物类似物非常相似，但与酰胺类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。研究了甘露内酯(MLD)类似物对牛胰腺磷脂酶A2的化学修饰和失活作用。发现该酶的Lys-56被修饰，这种修饰是导致该酶失活的原因。研究了PC-PLC催化Lyso-PC水解酶的反应动力学参数对pH的影响。1/Km和k&lt；CAT&Gt；的pH依赖曲线分别发生了一次和三次转变。虽然以PC为底物也得到了类似的结果，但伴随Lyso-PC和PC结合的可电离基团的PK筛分程度不同。研究了蜡状芽孢杆菌鞘磷脂酶(SMase)及其突变体(D126G和D156G)催化HNP水解酶的动力学参数与pH的关系。结果表明，Asp-126参与了底物的结合和催化作用，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.