Wu HengLiang, Yu Le, Tong Yujin, Ge Aimin, Yau Shuehlin, Osawa Masatoshi, Ye Shen
Catalysis Research Center, Hokkaido University, Sapporo 001-0021, Japan.
Biochim Biophys Acta. 2013 Feb;1828(2):642-51. doi: 10.1016/j.bbamem.2012.09.010. Epub 2012 Sep 17.
Atomic force microscopy (AFM) is employed to reveal the morphological changes of the supported phospholipid bilayers hydrolyzed by a phospholipase A(2) (PLA(2)) enzyme in a buffer solution at room temperature. Based on the high catalytic selectivity of PLA(2) toward l-enantiomer phospholipids, five kinds of supported bilayers made of l- and D-dipalmitoylphosphatidylcholines (DPPC), including l-DPPC (upper leaflet adjacent to solution)/l-DPPC (bottom leaflet) (or l/l in short), l/d, d/l, d/d, and racemic ld/ld, were prepared on a mica surface in gel-phase, to explicate the kinetics and mechanism of the enzyme-induced hydrolysis reaction in detail. AFM observations for the l/l bilayer show that the hydrolysis rate for l-DPPC is significantly increased by PLA(2) and most of the hydrolysis products desorb from substrate surface in 40 min. As d-enantiomers are included in the bilayer, the hydrolysis rate is largely decreased in comparison with the l/l bilayer. The time used to hydrolyze the as-prepared bilayers by PLA(2) increases in the sequence of l/l, l/d, ld/ld, and d/l (d/d is inert to the enzyme action). d-enantiomers in the enantiomer hybrid bilayers remain on the mica surface at the end of the hydrolysis reaction. It was confirmed that the hydrolysis reaction catalyzed by PLA(2) preferentially occurs at the edges of pits or defects on the bilayer surface. The bilayer structures are preserved during the hydrolysis process. Based on these observations, a novel kinetics model is proposed to quantitatively account for the PLA(2)-catalyzed hydrolysis of the supported phospholipid bilayers. The model simulation demonstrates that PLA(2) mainly binds with lipids at the perimeter of defects in the upper leaflet and leads to a hydrolysis reaction, yielding species soluble to the solution phase. The lipid molecules underneath subsequently flip up to the upper leaflet to maintain the hydrophilicity of the bilayer structure. Our analysis shows that d-enantiomers in the hybrid bilayers considerably reduce the hydrolysis rate by its ineffective binding with PLA(2).
原子力显微镜（AFM）用于揭示在室温缓冲溶液中，磷脂酶A(2)（PLA(2)）水解支撑磷脂双层的形态变化。基于PLA(2)对L-对映体磷脂的高催化选择性，在云母表面制备了五种由L-和D-二棕榈酰磷脂酰胆碱（DPPC）制成的支撑双层，包括L-DPPC（与溶液相邻的上层小叶）/L-DPPC（下层小叶）（简称为L/L）、L/D、D/L、D/D和外消旋LD/LD，处于凝胶相，以详细阐明酶促水解反应的动力学和机制。对L/L双层的AFM观察表明，PLA(2)显著提高了L-DPPC的水解速率，且大部分水解产物在40分钟内从底物表面解吸。当双层中包含D-对映体时，与L/L双层相比，水解速率大幅降低。PLA(2)水解所制备双层的时间按L/L、L/D、LD/LD和D/L的顺序增加（D/D对酶作用无反应）。对映体混合双层中的D-对映体在水解反应结束时仍保留在云母表面。证实了PLA(2)催化的水解反应优先发生在双层表面的凹坑或缺陷边缘。水解过程中双层结构得以保留。基于这些观察结果，提出了一个新的动力学模型来定量解释PLA(2)催化的支撑磷脂双层的水解。模型模拟表明，PLA(2)主要与上层小叶缺陷周边的脂质结合并引发水解反应，产生可溶于溶液相的物质。随后下面的脂质分子翻转到上层小叶以维持双层结构的亲水性。我们的分析表明，混合双层中的D-对映体因其与PLA(2)的无效结合而大大降低了水解速率。
