STEREOSPECIFICITY OF THE INTERACTION OF PORCINE PANCREATIC PHOSPHOLIPASE-A(2) WITH MICELLAR AND MONOMERIC INHIBITORS - A TIME-RESOLVED FLUORESCENCE STUDY OF THE TRYPTOPHAN RESIDUE

The binding effect of enantiomeric substrate analogs under micellar fo rm on the local conformation and dynamics of the N-terminal region of porcine pancreas phospholipase A2 was examined by time-resolved fluore scence measurements of its single tryptophan residue (Trp3). The compl exity of the fluorescence intensity decay of the unliganded protein (f our excited-state lifetime populations) suggests a conformational hete rogeneity of the N-terminal region of the protein. A considerable simp lification of the excited-state lifetime profile was specifically obse rved in the complex with one of the stereoisomers [(R)-2-tetradecanoyl amino)-hexanol-phosphocholine] at low inhibitor/protein molar ratio of almost-equal-to 9. This indicates the existence of a definite conform ation of the N-terminal region of the protein in the complex. No effec t was detected for the S-enantiomer. In parallel, the rotational mobil ity of the Trp residue in the complex with the R-enantiomer was reduce d. At a higher inhibitor/protein molar ratio of almost-equal-to 130, t he stereospecificity of the interaction was lost and complexes were fo rmed with both stereoisomers. These complexes were, however, not simil ar to the specific one either in terms of the local Trp3 environment o r of the volume of the rotating unit. The local effects of low amounts of monomeric inhibitors added to a preformed protein/micelle complex of a phospholipase A2 double mutant in which a Trp residue was genetic ally inserted near the active site at position 31 while the natural Tr p3 was replaced by Phe [Kuipers, O., Vincent, M., Brochon, J. C., Verh eij, H. M., de Haas, G. H. & Gallay, J. (1991) Biochemistry 30, 8771-8 785], were also monitored by time-resolved fluorescence of this single Trp residue. A stereospecific dependence of the local perturbations w as again observed. These results support the idea that the active conf ormation of the protein is reached in solution only after formation of a ternary complex: protein-interface-inhibitor.