Recombinant Human Acetylcholinesterase - Enzyme Engineering.
Abstract
Combination of recombinant DNA technology, kinetic studies and molecular modeling, are being employed in the design of novel biocatalysts with potential pharmacological use against organophosphate (OP) poisoning. Initial efforts were directed towards unravelling the intricate architecture of the catalytic machinery of human acetylcholinesterase (HuAChE). Thus, analysis of over 40 site directed mutants of HuAChE identified: (a) The esteratic site - Ser2O3, His447 and Glu334 constitute the catalytic triad. Glu2O2 at the bottom of the active site 'gorge' acts as modulator of the catalytic activity. (b) The anionic subsite - Trp86 is the element involved in pi-cation interactions with the quaternary ammonium of ACh (c) The acyl pocket - Phe295 and Phe297 determine specificity towards alkyl moiety of different substrates and OP inhibitors. (d) The peripheral anionic site(s) - involvement of residues Trp286, Tyr341 at the rim of the active site 'gorge' and Asp74 at its entrance. Asp74 affects both the catalytic and the peripheral anion sites of AChE. The aromatic amino acids Trp286 and Tyr341, through Asp74 and Tyr337 (which is located close to the catalytic center) are involved in the relay of inhibition signals induced by substrate binding at the periphery. (e) There is also evidence for a cross talk between the peripheral anionic site and the anionic subsite Trp86. Through these and other studies that-identified involvement of additional HuAChE residues (e.g Asp95, Aspl75, Asp4O4, His432, Cys580) in folding, intramolecular salt bridges and catalytic subunit interactions we are beginning to generate novel AChE enzymes with increased catalytic efficiencies or increased affinities towards different ligands.
Document Details
- Document Type
- Technical Report
- Publication Date
- May 13, 1993
- Accession Number
- ADP008865
Entities
People
- A. Ordentlich
- A. Shafferman
- B. Velan
- C. Kronman
- D. Barak
Organizations
- Israel Institute for Biological Research