van den Berg B, Tessari M, de Haas G H, Verheij H M, Boelens R, Kaptein R
Center for Biomembranes and Lipid Enzymology, Utrecht University, The Netherlands.
EMBO J. 1995 Sep 1;14(17):4123-31. doi: 10.1002/j.1460-2075.1995.tb00086.x.
The lipolytic enzyme phospholipase A2 (PLA2) is involved in the degradation of high-molecular weight phospholipid aggregates in vivo. The enzyme has very high catalytic activities on aggregated substrates compared with monomeric substrates, a phenomenon called interfacial activation. Crystal structures of PLA2s in the absence and presence of inhibitors are identical, from which it has been concluded that enzymatic conformational changes do not play a role in the mechanism of interfacial activation. The high-resolution NMR structure of porcine pancreatic PLA2 free in solution was determined with heteronuclear multidimensional NMR methodology using doubly labeled 13C, 15N-labeled protein. The solution structure of PLA2 shows important deviations from the crystal structure. In the NMR structure the Ala1 alpha-amino group is disordered and the hydrogen bonding network involving the N-terminus and the active site is incomplete. The disorder observed for the N-terminal region of PLA2 in the solution structure could be related to the low activity of the enzyme towards monomeric substrates. The NMR structure of PLA2 suggests, in contrast to the crystallographic work, that conformational changes do play a role in the interfacial activation of this enzyme.
脂解酶磷脂酶A2（PLA2）参与体内高分子量磷脂聚集体的降解。与单体底物相比，该酶对聚集底物具有非常高的催化活性，这种现象称为界面激活。有无抑制剂存在时PLA2的晶体结构相同，由此得出结论，酶的构象变化在界面激活机制中不起作用。使用双标记的13C、15N标记蛋白，通过异核多维NMR方法测定了溶液中游离的猪胰PLA2的高分辨率NMR结构。PLA2的溶液结构与晶体结构存在重要偏差。在NMR结构中，Ala1的α-氨基无序，涉及N端和活性位点的氢键网络不完整。在溶液结构中观察到的PLA2 N端区域的无序可能与该酶对单体底物的低活性有关。与晶体学研究相反，PLA2的NMR结构表明，构象变化在该酶的界面激活中确实起作用。
