Complex formation between the purified acetylcholinesterase described in the preceding communication and a series of bisquaternany inhibitors containing a benzoquinone group between two quaternary ammonium moieties maximally positioned 14 A apart was investigated by fluorescence quenching. The benzoquinone group absorbs maximally at 340 nm, providing optimal spectral overlap with the donor tryptophanyl residues in the protein. For the series of bisquaternary ligands, the complexes exhibit 44-49% of the fluorescence of the free enzyme and the dissociation constants vary from 2.8 x 10-8 to 1.2 x 10-5 M, depending on the substituents around the quaternary ammonium group. Uniform quenching of tryptophanyl fluorescence in each of the four binding sites on the tetrameric acetylcholinesterase molecule is observed, which indicates that the interbinding site distance exceeds 40-50 A. Back-titration with a nonquenching inhibitor, decamethonium, effects a return of the fluorescence to that of the free enzyme and permits a calculation of the affinity of a competing ligand. The catalytically participating serine in acetylcholinesterase has been esterified, forming the methanesulfonyl-, ethanesulfonyl-, propanesulfonyl-, diethoxyphosphoryl-, and diisopropoxyphosphoryl-enzymes. The affinity of three bisquaternary ligands possessing different bulk around the ammonium groups has been determined for the modified and catalytically inactive enzymes. These findings have provided a more detailed description of the topographical relationship between the sites of inhibitor binding and the active site. The specificity of binding for the bisquaternary ligand resides largely in the anionic subsite within the active site. In the modified enzymes the sulfone and phosphoryl oxygens can be expected to hydrogen bond in the oxyanion hole of the esteratic site, providing a discrete orientation for the alkoxy groups connected to the phosphorus and two possible orientations for the alkyl group adjoining the sulfonyl moiety. Binding affinity measurements enable a prediction of the favored orientation of the alkyl group with respect to the active center.
ACKNOWLEDGMENT We wish to thank Dr. J. J. Birktoft, Department of Chemistry, University of California, San Diego, for valuable discussions and for providing us with the active site tripeptide model taken from the crystallographic structure of chymotrypsin.
- Copyright ©, 1974, by Academic Press, Inc.