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

1. 采用pH-stat法研究了在不存在碳酰胺型、唑烷酮型和磺酸酰胺型底物类似物的情况下，Ca^<2+>和pH对I和II类磷脂酶A_2 （PLA_2s）催化的单分散1,2-二己醇- asn -甘油-3-磷酸胆碱水解动力学参数的影响。催化基团His 48对两种PLA_2s结合的Ca^<2+>依赖性和参与程度与恶唑烷酮型底物类似物非常相似，但与碳酰胺型和磺酸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。利用MLD -类似物研究了I族和II族PLA_2s的化学改性和失活。发现牛胰腺PLA_2s的Lys-56被mld类似物修饰，这种修饰与酶失活有关。结果表明，胰腺和南芥PLA_2s的失活源于界面识别位点的Lys残基的修饰，南芥、黄毒霉和russelli russelli的PLA_2s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。假定A.halys blomhoffii PLA_2s的失活是由于上述两个位点外的Lys残基修饰所致。我们认为上述两个位点外的Lys残基的修饰引起构象变化导致失活

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