Ludescher R D, Johnson I D, Volwerk J J, de Haas G H, Jost P C, Hudson B S
Department of Chemistry, University of Oregon, Eugene 97403.
Biochemistry. 1988 Aug 23;27(17):6618-28. doi: 10.1021/bi00417a061.
The rotational dynamics of the single tryptophan of porcine pancreatic phospholipase A2 and its zymogen (prophospholipase A2) have been studied by polarized fluorescence using steady-state and time-resolved single-photon counting techniques. The motion of Trp-3 in phospholipase A2 consists of a rapid subnanosecond wobble of the indole ring with an amplitude of about +/- 20 degrees accompanied by slower isotropic rotation of the entire protein. The rotational correlation times for overall particle rotational diffusion are consistent with conventional hydrodynamic theory. When phospholipase A2 binds to micelles of n-hexadecylphosphocholine, the amplitude of the fast ring rotation decreases. The whole particle rotational correlation time of the enzyme/micelle complex is smaller than the minimum value calculated from hydrodynamic theory. A similar result is obtained for the micelle itself by using the lipophilic probe transparinaric acid. These low values for the particle correlation times can be understood by postulating that an isotropic motion of the fluorophore in the small detergent particles contributes to the angular reorientation of the fluorophore. The internal reorientational motion of the tryptophan in the zymogen, prophospholipase A2, is of larger amplitude than that observed for the enzyme; specifically, the proenzyme exhibits a motion with a significant amplitude on the nanosecond time scale. This additional freedom of motion is attributed to segmental mobility of the N-terminal residues of prophospholipase A2. This demonstrates that this region of the protein is flexible in the zymogen but not in the processed enzyme. The implications of these findings for the mechanism of surface activation of phospholipase A2 are discussed by analogy with a trypsinogen-trypsin activation model.
运用稳态和时间分辨单光子计数技术，通过偏振荧光研究了猪胰磷脂酶A2及其酶原（前磷脂酶A2）中单个色氨酸的旋转动力学。磷脂酶A2中Trp-3的运动包括吲哚环快速的亚纳秒级摆动，摆动幅度约为±20度，同时伴随着整个蛋白质较慢的各向同性旋转。整体粒子旋转扩散的旋转相关时间与传统流体动力学理论一致。当磷脂酶A2与正十六烷基磷酰胆碱胶束结合时，快速环旋转的幅度减小。酶/胶束复合物的整体粒子旋转相关时间小于根据流体动力学理论计算出的最小值。通过使用亲脂性探针透明纳瑞酸，在胶束本身也得到了类似结果。粒子相关时间的这些低值可以通过假设荧光团在小洗涤剂颗粒中的各向同性运动有助于荧光团的角重排来理解。酶原前磷脂酶A2中色氨酸的内部重排运动幅度比在酶中观察到的要大；具体而言，前酶在纳秒时间尺度上表现出具有显著幅度的运动。这种额外的运动自由度归因于前磷脂酶A2 N端残基的片段流动性。这表明蛋白质的该区域在酶原中是灵活的，但在加工后的酶中不是。通过与胰蛋白酶原 - 胰蛋白酶激活模型类比，讨论了这些发现对磷脂酶A2表面激活机制的影响。
