Open AccessStereospecificity of the interaction of porcine pancreatic phospholipase A 2 with micellar and monomeric inhibitors
Author(s) -
VINCENT Michel,
DEVEER AnneMieke,
HAAS Gerard H.,
VERHEIJ Hubertus M.,
GALLAY Jacques
Publication year - 1993
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1993.tb18062.x
Subject(s) - chemistry , enantiomer , stereochemistry , monomer , residue (chemistry) , stereospecificity , tryptophan , micelle , fluorescence , phospholipase a2 , enzyme , biochemistry , amino acid , organic chemistry , aqueous solution , physics , quantum mechanics , catalysis , polymer
The binding effect of enantiomeric substrate analogs under micellar form on the local conformation and dynamics of the N‐terminal region of porcine pancreas phospholipase A 2 was examined by time‐resolved fluorescence measurements of its single tryptophan residue (Trp3). The complexity of the fluorescence intensity decay of the unliganded protein (four excited‐state lifetime populations) suggests a conformational heterogeneity of the N‐terminal region of the protein. A considerable simplification of the excited‐state lifetime profile was specifically observed in the complex with one of the stereoisomers [( R )‐2‐tetradecanoylamino)‐hexanol‐phosphocholine] at low inhibitor/protein/molar ratio of ∼ 9. This indicates the existence of a definite conformation of the N‐terminal region of the protein in the complex. No effect was detected for the S ‐enantiomer. In parallel, the rotational mobility of the Trp residue in the complex with the R ‐enantiomer was reduced. At a higher inhibitor/protein molar ratio of ∼ 130, the stereospecificity of the interaction was lost and complexes were formed with both stereoisomers. These complexes were, however, not similar to the specific one either in terms of the local Trp3 environment or 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 A 2 double mutant in which a Trp residue was genetically inserted near the active site at position 31 while the natural Trp3 was replaced by Phe [Kuipers, O., Vincent, M., Brochon, J. C., Verheij, H. M., de Haas, G. H. & Gallay, J. (1991) Biochemistry 30, 8771–8785], were also monitored by time‐resolved fluorescence of this single Trp residue. A stereospecific dependence of the local perturbations was again observed. These results support the idea that the active conformation of the protein is reached in solution only after formation of a ternary complex: protein‐interface‐inhibitor.