Acetylcholine hydrolysis by acetylcholinesterase is inhibited at high substrate concentrations. To determine the residues involved in this phenomenon, we have mutated most of the residues lining the active-site gorge but mutating these did not completely eliminate hydrolysis. Thus, we analyzed the effect of a nonhydrolysable substrate analogue on substrate hydrolysis and on reactivation of an analogue of the acetylenzyme. Analyses of various models led us to propose the following sequence of events: the substrate initially binds at the rim of the active-site gorge and then slides down to the bottom of the gorge where it is hydrolyzed. Another substrate molecule can bind to the peripheral site: (a) when the choline is still inside the gorge -it will thereby hinder its exit; (b) after choline has dissociated but before deacetylation occurs -binding at the peripheral site increases deacetylation rate but (c) if a substrate molecule bound to the peripheral site slides down to the bottom of the activesite before the catalytic serine is deacetylated, its new position will prevent the approach of water, thus blocking deacetylation.

A major result of incoherent elastic neutron-scattering experiments on protein powders is the strong dependence of the intramolecular dynamics on the sample environment. We performed a series of incoherent elastic neutron-scattering experiments on lyophilized human butyrylcholinesterase (HuBChE) powders under different conditions (solvent composition and hydration degree) in the temperature range from 20 to 285 K to elucidate the effect of the environment on the enzyme atomic mean-square displacements. Comparing D(2)O- with H(2)O-hydrated samples, we were able to investigate protein as well as hydration water molecular dynamics. HuBChE lyophilized from three distinct buffers showed completely different atomic mean-square displacements at temperatures above approximately 200 K: a salt-free sample and a sample containing Tris-HCl showed identical small-amplitude motions. A third sample, containing sodium phosphate, displayed highly reduced mean-square displacements at ambient temperature with respect to the other two samples. Below 200 K, all samples displayed similar mean-square displacements. We draw the conclusion that the reduction of intramolecular protein mean-square displacements on an Angstrom-nanosecond scale by the solvent depends not only on the presence of salt ions but also on their type.

Insect acetylcholinesterase (AChE), an enzyme whose catalytic site is located at the bottom of a gorge-like structure, hydrolyzes its substrate over a wide range of concentrations (from 2 M to 300 mM). AChE is activated at low substrate concentrations and inhibited at high substrate concentrations. Several rival kinetic models have been developed to try to describe and explain this behavior. One of these models assumes that activation at low substrate concentrations partly results from an acceleration of deacetylation of the acetylated enzyme. To test this hypothesis, we used a monomethylcarbamoylated enzyme, which is considered equivalent to the acylated form of the enzyme and a non-hydrolyzable substrate analog, 4-oxo-N,N,N-trimethylpentanaminium iodide. It appears that this substrate analog increases the decarbamoylation rate by a factor of 2.

Incoherent elastic neutron scattering experiments on members of the cholinesterase family were carried out to investigate how molecular dynamics is affected by covalent inhibitor binding and by differences in primary and quaternary structure. Tetrameric native and soman-inhibited human butyrylcholinesterase (HuBChE) as well as native dimeric Drosophila melanogaster acetylcholinesterase (DmAChE) hydrated protein powders were examined. Atomic mean-square displacements (MSDs) were found to be identical for native HuBChE and for DmAChE in the whole temperature range examined, leading to the conclusion that differences in activity and substrate specificity are not reflected by a global modification of subnanosecond molecular dynamics. MSDs of native and soman-inhibited HuBChE were identical below the thermal denaturation temperature of the native enzyme, indicating a common mean free-energy surface. Denaturation of the native enzyme is reflected by a relative increase of MSDs consistent with entropic stabilization of the unfolded state. The results suggest that the stabilization of HuBChE phosphorylated by soman is due to an increase in free energy of the unfolded state due to a decrease in entropy.