Burack W R, Gadd M E, Biltonen R L
Department of Biochemistry, University of Virginia Health Sciences Center, Charlottesville 22908, USA.
Biochemistry. 1995 Nov 14;34(45):14819-28. doi: 10.1021/bi00045a024.
Phospholipase A2-catalyzed hydrolysis of vesicular phospholipid has been used to model the modulation of an enzyme's function by membrane properties. Phospholipase A2's (PLA2) kinetics toward large unilamellar vesicles (LUV) composed of dipalmitoylphosphatidylcholine (DPPC) are anomalous; these is a slow initial phase of catalysis (a lag) which ends abruptly with a sudden increase in the catalytic rate (a burst). The sudden increase in activity due to the accumulation of a critical mole fraction of reaction products and substrate undergoes compositional phase separation. In this work, we address the molecular details of the coupling between compositional phase separation and activation of PLA2. A prominent model for this coupling is that compositional phase separation leads to a surface for which PLA2 has increased affinity, resulting in the recruitment of PLA2 from solution to the surface. Here, we show that the bulk of PLA2 is associated with the membrane at a time well before the abrupt increase in catalytic rate. This finding indicates that there must be a relatively inactive, membrane-bound state. Furthermore, PLA2's kinetics are anomalous even when the substrate comprises a surface to which PLA2 is bound throughout the time course. With DPPC LUV as the substrate, detailed time courses show that the description of the time course as a lag and a burst is inadequate. Instead, the time course consists of multiple phases of acceleration and deceleration. The data presented here suggest that all these various changes in catalytic rate may be due to product-induced changes in membrane properties. In particular, we suggest that nonequilibrium, microheterogeneities of lipid composition may underlie these very complicated kinetics.
磷脂酶A2催化的囊泡磷脂水解已被用于模拟膜性质对酶功能的调节。磷脂酶A2（PLA2）对由二棕榈酰磷脂酰胆碱（DPPC）组成的大单层囊泡（LUV）的动力学是异常的；催化作用有一个缓慢的初始阶段（滞后），该阶段突然结束，催化速率突然增加（爆发）。由于反应产物和底物的临界摩尔分数积累导致的活性突然增加，底物经历了组成相分离。在这项工作中，我们研究了组成相分离与PLA2激活之间耦合的分子细节。这种耦合的一个突出模型是，组成相分离导致了一个表面，PLA2对其亲和力增加，从而导致PLA2从溶液中募集到表面。在这里，我们表明，在催化速率突然增加之前的一段时间，大部分PLA2就已与膜结合。这一发现表明，必然存在一种相对无活性的膜结合状态。此外，即使底物在整个时间进程中都包含PLA2结合的表面，PLA2的动力学也是异常的。以DPPC LUV为底物，详细的时间进程表明，将时间进程描述为滞后和爆发是不够的。相反，时间进程由加速和减速的多个阶段组成。这里给出的数据表明，催化速率的所有这些不同变化可能是由于产物诱导的膜性质变化。特别是，我们认为脂质组成的非平衡微不均匀性可能是这些非常复杂的动力学的基础。
