Ranganathan Radha, Tcacenco Celize Maia, Rosseto Renato, Hajdu Joseph
Department of Physics and Astronomy and Center for Supramolecular Studies, California State University, Northridge, CA 91330-8268, USA.
Biophys Chem. 2006 Jul 20;122(2):79-89. doi: 10.1016/j.bpc.2006.02.012. Epub 2006 Mar 23.
Phospholipase C catalyzed hydrolysis of dimyristoyl phosphatidylcholine (DMPC) in phospholipid-bile salt mixed micelles was studied with particular attention on the relationship between interfacial enzyme activity and the physicochemical properties of substrate aggregates. Steady state kinetics is observed and it is argued that conditions for steady state exist because the enzyme encounters a steady supply of substrate by hopping between micelles at a rate faster than the chemical reaction rate. An existing kinetic model is reformulated to a more usable form. This presents a new approach to treating the kinetic data and allows extraction of the kinetic parameters of the model from the activity dependence on micellar lipid substrate surface concentration. The kinetic parameters were found to depend on the physicochemical properties of substrate aggregates, but remain constant over a range of lipid and bile salt concentrations. The substrate aggregates were characterized by time-resolved fluorescence quenching (TRFQ). The activity values and the micelle sizes group into two sets: (i) larger micelles for bile salt/lipid <or=5 showing higher activity and shorter steady state duration (<or=4 min) and (ii) smaller micelles for bile salt/lipid > 5 with lower activity and longer steady state ( approximately 10 min). At least two sets of parameters, for bile salt/lipid <or=5 and >5, characterize the kinetics. Higher enzyme-micelle dissociation constant and lower catalytic rate are found for the group of smaller micelles. An explanation supporting our finding is that as micelles become smaller the overlap area for enzyme-micelle binding decreases, leading to weaker binding. Consequently the enzyme dissociation constant increases. Extension of the present approach to other phospholipases and substrates to establish its generality and correlation between micelle size and the catalytic rate are areas for future investigations.
研究了磷脂酶C催化磷脂 - 胆盐混合胶束中二肉豆蔻酰磷脂酰胆碱（DMPC）的水解反应，特别关注界面酶活性与底物聚集体物理化学性质之间的关系。观察到了稳态动力学，并且认为存在稳态条件是因为酶通过以比化学反应速率更快的速率在胶束之间跳跃而遇到稳定的底物供应。将现有的动力学模型重新表述为更实用的形式。这提出了一种处理动力学数据的新方法，并允许从活性对胶束脂质底物表面浓度的依赖性中提取模型的动力学参数。发现动力学参数取决于底物聚集体的物理化学性质，但在一定范围的脂质和胆盐浓度内保持恒定。通过时间分辨荧光猝灭（TRFQ）对底物聚集体进行了表征。活性值和胶束大小分为两组：（i）胆盐/脂质≤5时较大的胶束，显示出较高的活性和较短的稳态持续时间（≤4分钟），以及（ii）胆盐/脂质> 5时较小的胶束，活性较低且稳态较长（约10分钟）。至少有两组参数，分别对应胆盐/脂质≤5和> 5的情况，表征了动力学。对于较小胶束组，发现酶 - 胶束解离常数较高且催化速率较低。支持我们这一发现的一种解释是，随着胶束变小，酶 - 胶束结合的重叠面积减小，导致结合变弱。因此酶解离常数增加。将本方法扩展到其他磷脂酶和底物以确定其通用性以及胶束大小与催化速率之间的相关性，是未来研究的方向。
