Mutation Report for: F295A/F338A/V407F_human-ACHE

Determination of the 3D-structure of acetylcholinesterase (AChE) of Torpedo californica over a decade ago, and more recently that of human enzyme together with extensive targeted mutagenesis of the mammalian AChEs led to a fine mapping of the multiple functional domains within the active center of the enzyme. Many of the contributions of this active center architecture to accommodation of noncovalent ligands could be deduced from the X-ray structures of the corresponding HuAChE complexes. Yet, Michaelis complexes leading to transient covalent adducts are not amenable to structural analysis. Since the rates of formation of the covalent adducts depend predominantly on the stabilities of the corresponding Michaelis complexes, it is essential to characterize the specific interactions contributing to stabilization of these complexes. Functional analysis of interactions with HuAChE enzymes allows for such characterization for carbamates, like pyridostigmine or rivastigmine, much in the same way as that for the noncovalent therapeutic ligands nivalin or aricept. In fact, the observed differences between the affinities toward carbamates and the noncovalent ligands seem to result from specific structural characteristics of the inhibitors rather than from the decomposition path of the particular complex. Replacements at the cation binding site (Trp86), hydrogen bond network (Glu202, Tyr133, Glu450), and hydrophobic pocket result in similar effects for the covalent as well as for the noncovalent inhibitors. Also, while the effects of perturbing the aromatic trapping of the catalytic His447 for pyridostigmine and nivalin were analogous to those for the substrate, the corresponding effects for rivastigmine and aricept were quite different. Thus, elucidation of the functional architecture of the HuAChE active center is bound to be of considerable utility in the current effort to design novel covalent AChE inhibitors as therapeutics for Alzheimer's disease (AD).

While substitution of the aromatic residues (Phe295, Phe338), located in the vicinity of the catalytic His447 in human acetylcholinesterase (HuAChE) had little effect on catalytic activity, simultaneous replacement of both residues by aliphatic amino acids resulted in a 680-fold decrease in catalytic activity. Molecular simulations suggested that the activity decline is related to conformational destabilization of His447, similar to that observed for the hexamutant HuAChE which mimics the active center of butyrylcholinesterase. On the basis of model structures of other cholinesterases (ChEs), we predicted that catalytically nonproductive mobility of His447 could be restricted by introduction of aromatic residue in a different location adjacent to this histidine (Val407). Indeed, the F295A/F338A/V407F enzyme is 170-fold more reactive than the corresponding double mutant and only 3-fold less reactive than the wild-type HuAChE. However, analogous substitution of Val407 in the hexamutant HuAChE (generating the heptamutant Y72N/Y124Q/W286A/F295L/F297V/Y337A/V407F) did not enhance catalytic activity. Reactivity of these double, triple, hexa, and hepta mutant HuAChEs was monitored toward covalent ligands such as organophosphates and the transition state analogue TMFTA, which probe, respectively, the facility of the enzymes to accommodate Michaelis complexes and to undergo the acylation process. The findings suggest that in the F295A/F338A mutant the two His447 conformational states, which are essential for the different stages of the catalytic process, seem to be destabilized. On the other hand, in the F295A/F338A/V407F mutant only the state involved in acylation is impaired. Such differential effects on the His447 conformational properties demonstrate the general role of aromatic residues in cholinesterases, and probably in other serine hydrolases, in "trapping" of the catalytic histidine and thereby in optimization of catalytic activity.