New Inhibitors of the Peripheral Site in Acetylcholinesterase that Specifically Block Organophosphorylation
Abstract
Examination of the enzyme structure for acetylcholinesterase (AChE) reveals two sites of ligand interaction: The peripheral site (P-site) located at the entrance of the gorge, and the acylation site (A-site) at the base of the gorge. Our goal is to develop high affinity cyclic peptide ligands specific for the P-site while allowing the passage of acetylcholine to the A-site for use by personnel at risk for nerve gas exposure. Our strategy involves the covalent tethering of cyclic inhibitors via a methanethiosulfonate (MTS) linkage to a cysteine on the AChE mutant, H287C. To validate this approach we used cat ionic ligands with demonstrated affinity for the AChE A-site that are attached to MTS tethers of various lengths. After labeling we separated modified from unmodified enzymes using affinity chromatography on acridinium resin. Enzymes modified with ligands having a significant effect on the A-site elute in a NaCl wash, while unmodified enzyme elutes only with the AChE inhibitor decamethonium. We compared the substrate dependence of hydrolysis rates on the substrate concentration for unmodified H287C as well as 3 modified enzymes and found that 2 of the modified enzymes shifted the hydrolysis curve, with large increases in K(app) and K(12) Selective inhibition by propidium at the P-site and tacrine at the A-site measure the effect of labeling. We compared K(12) values and found that the shortest tethers had little effect on K(12) Medium length tethers interfered with propidium binding to the P-site while K(12) values for tacrine inhibition increased progressively as the size of the tethered ligand increases.
Document Details
- Document Type
- Technical Report
- Publication Date
- Apr 01, 2003
- Accession Number
- ADA417604
Entities
People
- Terrone L. Rosenberry
Organizations
- Mayo Clinic