| Summary: | 博士 === 國立中正大學 === 化學所 === 98 === Single molecule fluorescence microscopy can reveal individual enzyme actions underlying a broad distribution of enzyme activities in enzymology. The mechanism of gel-state biological model membranes degraded by phospholipase A2 (PLA2) had been suggested to follow a scooting mode based on the results of the atomic force microscopy (AFM). However, the direct contact of the AFM tip with membrane surface strongly perturbs the surface properties of lipid membrane. Herein, employment of a method which can directly watch single dye-labeled PLA2 molecules could give new insights to the hydrolysis mechanisms. By tracking the spatial distributions and diffusion motions of single PLA2 molecules on defect contained membranes, we found that PLA2s bound to the boundaries around membrane defects with an initial low-activity period (lag-phase), followed by a burst of PLA2s binding on newly created active sites. Besides, those PLA2 bound after lag-burst period showed immobilized under the ~40 nm spatial resolving power of our detection platform. By direct watching the enzyme motions at single molecule level, a relay model is proposed where the reaction is activated by the initial bound PLA2s which in turn produce composition defects allowing more PLA2s docking to further hydrolyze membrane substrate.
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