Molecular Theory of the Catalytic Function of Phospholipase A2

磷脂酶A2催化功能的分子理论

基本信息

批准号：
02671022
负责人：
IKEDA Kiyoshi
金额：
$ 1.66万
依托单位：
Osaka University of Pharmaceutical Science
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1992
项目状态：
已结题

项目摘要

1. Kinetics of the hydrolysis of monodispersed and micellar sustrates, catalyzed by Group I or II phospholipase A_2 (PLA_2s) in the presence of a saturating Ca^<2+> concentration indicated that deprotonated state of the catalytic group His 48 and protonated state of Tyr 52, the site of which is located in close proximity to the imidazole ring of His 48 were found to be essential for the catalysis. The importance of an ionized state of the N-terminal alpha-amino group at the active site was also indicated for Group I enzymes. The pK values of both His 48 and Tyr 52 of the enzyme-Ca^<2+> complex shifted markedly to the alkaline side on binding to micellar substrates, whereas no significant pK shifts were noted for the bindings to monodispersed substrates.2. Kinetic studies showed that Ca^<2+> binding to the both types of PLA_2s was essential for the catalysis. Substrate bindings to Group I PLA_2s were independent of the Ca^<2+> binding, whereas those for Group II enzymes were facilitated … More more than 10 times by the Ca^<2+> binging. The latter result was compatible with the hypothesis that an intermediate complex would be stabilized by coordination of the bound Ca^<2+> ion with the phosphoryl group and the carbonyl group at the sn-2 position of the substrate molecule. However, the former, the former result for Group I enzymes seemed incompatible for this mechanism. The X-ray crystallographic studies on the bindings of a substrate analog having an amide-bond instead of the sn-2 ester bond, dodecanoyl-2-aminohexanol-1-phosphoglycol, indicated that the carbonyl oxygen and phosphoryl moiety interact directly with the bound Ca^<2+> ion at the substrate binding site, suggesting that the binding of this analog should depend on the Ca^<2+> binding. Our studies in solution to confirm this showed the binding constants of this analog to both types of enzymes were increased significantly as the degrees of Ca^<2+> bindings increase, indicating that the structures of enzyme-substrate analog complex and enzyme-Ca^<2+>-analog complex in solution are very similar to each other as those in crystal and that the structures of the enzyme-analog complexes are stabilized by the Ca^<2+> binding. The enzyme-Ca^<2+>-genuine substrate complexes for the both types of PLA_2s are also considered to have similar structures.3. Some metal ions including lantanide ions were showed to bind to PLA_2s and to their genuine-substrate complexes in a manner similar to that of Ca^<2+> ion and to produce the PLA_2 activites (less than 25%). Less

1。指示的评分Ca^<2+>浓度指示指示的催化群的印度状态他的48和Tyr 52的质子化状态，该地点位于靠近其48的咪唑环的附近，这对于催化是必不可少的。对于与单分散的底物的结合，2。比Ca^<2+> 10次，后者的结果与以下假设兼容，即中间体将通过与磷酸基组的结合Ca^<2+>离子的协调来稳定。但是，底物分子。磷酸二醇在底物结合位点表明羰基氧和磷酸化与结合的Ca^<2+>离子，这表明类似物的结合应依赖于Ca^<2+>我们在溶液中的研究以确认结合的结合随着Ca^<2+>结合的程度增加，两种类型的ES的常数都显着增加，指示了酶 - 底物类似物的酶类似物的融合和酶-Ca^2+>酶的酶，以及晶体中的酶的结构以及酶的结构 - nnog络合物通过Ca^ <2+>结合稳定。以类似于Ca^<2+>离子的方式与PLA_2S及其真正的底物复合物结合并产生PLA_2 Activites（较少的Toan 25％）。