Jain M K, Ranadive G, Yu B Z, Verheij H M
Department of Chemistry, University of Delaware, Newark 19716.
Biochemistry. 1991 Jul 23;30(29):7330-40. doi: 10.1021/bi00243a038.
Interfacial catalysis in the scooting mode by phospholipase A2 (PLA2) from pancreas and venoms (18 different preparations) was examined on vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol under the conditions where the rates of transbilayer and intervesicle exchanges of the enzyme, substrate, and the products of hydrolysis were negligible on the time scale (less than 30 min) on which all the substrate molecules on the outer monolayer of the target vesicles were hydrolyzed. The reaction progress curves for all PLA2s exhibited no latency period (less than 3 s). When the vesicle to PLA2 ratio in the reaction mixture was high so that according to the Poissonian distribution model at most one enzyme was bound to a vesicle, the extent of hydrolysis increased linearly with the amount of enzyme in the reaction mixture. However, the extent of hydrolysis per enzyme, NS, remained the same for all PLA2s, and it corresponded to the size of the target vesicles determined by independent methods. Similarly, the initial rate of hydrolysis increased linearly with the enzyme concentration, and the slope of the log-log plot was one under the conditions of one or more enzyme per vesicle. Such observations showed that monomeric PLA2 is fully catalytically active at the interface. This conclusion was supported by the absence of intermolecular resonance energy transfer from tryptophan-3 donor in the native PLA2 to the anthraniloyl acceptor in An87-PLA2, the catalytically active derivative of PLA2 with an anthraniloyl fluorophore on lysine 87. In this system, intermolecular resonance energy transfer was seen only when the donor-acceptor molecules were "crowded" at a high surface density with a relatively low lipid to protein mole ratio. On the basis of these results, it was concluded that secretory PLA2s from venoms and pancreas are fully catalytically active as monomers. Additional studies reported here showed that acylation of PLA2 was not necessary for catalysis or binding to the interface and that the binding of the substrate to the active site of PLA2 was not necessary for the binding of the enzyme to the interface.
在1,2-二肉豆蔻酰-sn-甘油-3-磷酸甲醇囊泡上，研究了胰腺和毒液来源的磷脂酶A2（PLA2，18种不同制剂）在“滑动”模式下的界面催化作用。实验条件为：在靶囊泡外单层上所有底物分子水解所需的时间尺度（小于30分钟）内，酶、底物和水解产物的跨膜和囊泡间交换速率可忽略不计。所有PLA2的反应进程曲线均无延迟期（小于3秒）。当反应混合物中囊泡与PLA2的比例较高时，根据泊松分布模型，最多一个酶分子结合到一个囊泡上，水解程度随反应混合物中酶量的增加呈线性增加。然而，所有PLA2的每个酶分子的水解程度NS保持不变，且与通过独立方法测定的靶囊泡大小相对应。同样，水解初始速率随酶浓度呈线性增加，在每个囊泡有一个或多个酶的条件下，对数-对数图的斜率为1。这些观察结果表明单体PLA2在界面处具有完全催化活性。这一结论得到以下结果的支持：在天然PLA2中，色氨酸-3供体与赖氨酸87上带有邻氨基苯甲酰荧光团的PLA2催化活性衍生物An87-PLA2中的邻氨基苯甲酰受体之间不存在分子间共振能量转移。在该系统中，只有当供体-受体分子以相对较低的脂质与蛋白质摩尔比“聚集”在高表面密度时，才会出现分子间共振能量转移。基于这些结果，得出结论：毒液和胰腺分泌的PLA2作为单体具有完全催化活性。此处报道的其他研究表明，PLA2的酰化对于催化或与界面结合不是必需的，并且底物与PLA2活性位点的结合对于酶与界面的结合也不是必需的。
