Rotational Dynamics of the Single Tryptophan of Porcine Pancreatic Phospholipase A₂, Its Zymogen, and an Enzyme/Micelle Complex: A Steady-State and Time-Resolved Anisotropy Study

The rotational dynamics of the single tryptophan of porcine pancreatic phospholipase A₂and 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 A₂consists of a rapid subnanosecond wobble of the indole ring with an amplitude of about ±20° 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 A₂binds 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 trans-parinaric 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 A₂are discussed by analogy with a trypsinogen-trypsin activation model.