Kinetics of binding of phospholipase A2 to lipid/water interfaces and its relationship to interfacial activation.
Kinetics of binding of phospholipase A2 to lipid/water interfaces and its relationship to interfacial activation.
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磷脂酶 A2 与脂质/水界面结合的动力学及其与界面活化的关系。
DOI：
10.1016/0005-2736(88)90007-7
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发表时间：
1988
期刊：
影响因子：
--
通讯作者：
DeHaas,GH
中科院分区：
文献类型：
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作者：
Jain,MK;Rogers,J;DeHaas,GH
磷脂相a2和磷脂酶a2与各种底物和不可水解磷脂类似物的囊泡和胶束结合的时间过程是通过监测蛋白质上的Trp-3或囊泡表面上的5-二甲氨基萘-1-磺酰基（丹酰）发色团的荧光强度变化来获得的。磷脂酶a2荧光强度的时间依赖性增加仅在催化和平衡结合也被观察到的条件下观察到。结合的总体动力学由两个速率常数描述。这两种蛋白质结合的快速二阶速率常数（k A）以磷脂为单体表示为2·10.7 / s / mol，以囊泡表示为10·10 / s / mol。这可能是蛋白质与囊泡作为结合的第一步的扩散受限遭遇。对于磷脂酶a2的结合，还发现了一个额外的一级速率常数（kb = 4 / s），但对于前磷脂期a2则没有。EGTA存在时，结合的磷脂酶a2的解吸速率非常慢（小于0.0002 / s），而结合的前磷脂酶a2的解吸速率要快得多（2.9 / s）。根据磷脂相a2和原磷脂酶a2的界面催化周转速率的差异，阐述了这些速率常数的机理意义。如其他地方所示（Jain等）。Biochim。Biophys。磷脂酶a2对阴离子囊泡的水解发生在滑动模式下，使得结合酶在目标囊泡上停留数千个催化周转循环。另一方面，如本文所示，磷脂酶a2水解的动力学主要是其囊泡间交换。因此，在跳跃模式下，磷脂酶a2的界面催化在每个循环中都有一个开启和一个关闭步骤，导致催化周转次数约为每秒1.2次。因此，从跳跃到滑行的催化模式的变化为磷脂酶a2激活界面催化提供了动力学基础。
The time-course of binding of phospholiphase A 2 and prophospholipase A 2 to vesicles and micelles of a variety of substrate and nonhydrolyzable phospholipid analogs is obtained by monitoring the change in the fluorescence intensity of Trp-3 on the protein or of the 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) chromophore on the surface of the vesicles. The time-dependent increase in the fluorescence intensity of phospholipase A 2 is observed only under conditions where catalysis and equilibrium binding are also observed. The overall kinetics of binding is described by two rate constants. A rapid second-order rate constant (k a) for binding of both the proteins is 2· 10 7 per s per mol expressed in terms of phospholipids as monomers, and 10 10 per s per mol expressed in terms of vesicles. This is probably a diffussion-limited encounter of the protein with vesicles as the first step in binding. An additional first-order rate constant (K b= 4 per s) was also discerned for the binding of phospholipase A 2 but not for prophospholiphase A 2. The rate of desorption of the bound phospholipase A 2 in the presence of EGTA is very slow (less than 0.0002 per s), whereas the rate of desorption of the bound prophospholipase A 2 is much more rapid (2.9 per s). The mechanistic significance of these rate constants is elaborated in terms of the differences in the rates of interfacial catalytic turnover of phospholiphase A 2 and prophospholipase A 2. As shown elsewhere (Jain et al. Biochim. Biophys. Acta 860, 435–447) the hydrolysis of anionic vesicles by phospholipase A 2 occurs in the scooting mode such that the bound enzyme remains on the target vesicles for several thousand catalytic turnover cycles. On the other hand, as shown in this paper, the kinetics of hydrolysis by prophospholipase A 2 is dominated by its intervesicle exchange. Therefore, interfacial catalysis by prophospholipase A 2 in the hopping mode would involve an on-and an off-step in each cycle, resulting in a catalytic turnover number of about 1.2 per s. A change from the hopping to the scooting mode of catalysis thus provides the kinetic basis for activation of interfacial catalysis by phospholipase A 2 compared to that for prophospholipase A 2.