Changes in Ca2+ affinity upon activation of Agkistrodon piscivorus piscivorus phospholipase A2.
Changes in Ca2+ affinity upon activation of Agkistrodon piscivorus piscivorus phospholipase A2.
复制标题
Agkistrodon piscivorus piscivorus 磷脂酶 A2 激活后 Ca2 亲和力的变化。
作者：
Lathrop,B;Gadd,M;Biltonen,RL;Rule,GS
用定点突变和荧光光谱分析的方法，研究了在磷脂酰胆碱囊泡表面磷脂酶A2激活过程中，钙离子对该酶亲和力的变化。在脂质结合和随后的激活过程中发生的荧光变化被归因于蛋白质中三个单独的Trp残基中的每一个。这是通过产生一组突变蛋白质来实现的，每个突变蛋白质都缺少一个或多个Trp残基。位于界面结合区的Trp21和Trp119在蛋白质与小的单层两性离子小泡或含有足够阴离子脂的大的单层小泡结合时都显示出荧光的变化。Trp31位于Ca~(2+)结合环附近，与脂双层结合时荧光变化不大。在酶的激活过程中，蛋白质的荧光也会发生变化。这些变化来自残基Trp31以及残基Trp21和Trp119。Trp31荧光变化的钙依赖性表明，酶对钙的亲和力在激活后至少增加了3个数量级。这些研究表明，要么是酶的构象发生了变化，要么是钙亲和力的增加反映了钙、酶和底物三元复合体的形成。
Changes in the affinity of calcium for phospholipase A2fromAgkistrodon piscivorus piscivorusduring activation of the enzyme on the surface of phosphatidylcholine vesicles have been investigated by site-directed mutagenesis and fluorescence spectroscopy. Changes in fluorescence that occur during lipid binding and subsequent activation have been ascribed to each of the three individual Trp residues in the protein. This was accomplished by generating a panel of mutant proteins, each of which lacks one or more Trp residues. Both Trp21, which is found in the interfacial binding region, and Trp119 show changes in fluorescence upon protein binding to small unilamellar zwitterionic vesicles or large unilamellar vesicles containing sufficient anionic lipid. Trp31, which is near the Ca2+binding loop, exhibits little change in fluorescence upon lipid bilayer binding. A change in the fluorescence of the protein also occurs during activation of the enzyme. These changes arise from residue Trp31 as well as residues Trp21 and Trp119. The calcium dependence of the fluorescence change of Trp31 indicates that the affinity of the enzyme for calcium increases at least 3 orders of magnitude upon activation. These studies suggest either that a change in conformation of the enzyme occurs upon activation or that the increase in calcium affinity reflects formation of a ternary complex of calcium, enzyme, and substrate.