Acetyicholinesterase (AChE) terminates synaptic transmission at cholinergic synapses by hydrolyzing the neurotransmitter acetyicholine. We will focus on molecular mechanisms responsible for the high catalytic efficiency of AChE. Such information may allow the design of new classes of drugs to protect against organophosphate toxicity. The structure of AChE reveals an active site gorge with two sites of ligand interaction separated by 12-15 A: an acylation site (or A site) at the base of the gorge, and a peripheral site (or P site) at its mouth. We discovered that substrates or inhibitors that bind to the P site of AChE impose a steric blockade on ligand entry to and exit from the A site. We now find that ligand binding to the P site also increases the acylation rate constant for a substrate bound to the A site, while binding of the neurotoxin fasciculin to the P site results in a dramatic decrease in these acylation rate constants. We will test a mechanistic model involving the P and A sites that can account for these features.
Our first aim examines thioflavin T, whose fluorescence is enhanced when it is bound to the P site and partially quenched when ligands bind to the A site to form a ternary complex, indicating a local conformational change. We will pursue crystal structure determinations of the AChE-thioflavin T complex with collaborators and use thioflavin T binding and fluorescence to monitor acceleration of organophosphorylation rate constants with wild type and mutant AChEs. In a new second aim, we will modify the SH groups of mutants E81C and E84C with a solvent-sensitive fluorophore and examine interactions with the P and A sites. We will ask whether ligand binding to the P site alone is sufficient to induce conformational movement of the peptide loop that contains these residues.
Our third aim i s to determine whether substrates closely analogous to acetyicholine can accelerate acylation reactions by binding to the P site. We will test our kinetic model by comparing predicted and measured P site substrate affinities for acetylthiocholine and m-(trimethylammonio)acetanilide in wild type, W86F and W86A AChEs.
Our fourth aim i s to identify the AChE residues through which fasciculin exerts an inhibitory conformational effect on the A site. Our hypothesis, that a segment of an Omega-loop (residues 72 to 87) is involved in transducing this effect, is supported by data showing loss of much of the inhibitory effect with the D74G and W86F mutants. To test this hypothesis further, we will construct additional site-specific AChE mutants as well as fasciculin mutants and determine whether these residues can be altered to retain inhibition of organophosphorylation but decrease inhibition of substrate hydrolysis.
Showing the most recent 10 out of 46 publications
