Ionic Hydrogen Bonds in Bioenergetics. 4
J. Am. Chem. Soc., Vol. 121, No. 7, 1999 1555
ACh,gt (Table 3). However, for some of the complexes, the
weakening of the O7‚‚‚H13 interaction is partially compensated
for by a strengthening of the O9‚‚‚H23 interaction. Another
manifestation of the effect of external solvation on internal
solvation is that the most stable ACh conformation is gg′ in
the isolated ion, whereas it is gg or gt in the cluster ions.
One way to determine how the intramolecular hydrogen
bonding influences ACh‚‚‚ligand interactions is to compare the
relative enthalpies of the ions with the ACh,tt rotamer to those
of the ions with the other rotamers (Tables 5 and 6). More
specifically, the enthalpy of ACh,tt is 13.0 kJ/mol (3.1 kcal/
mol) less stable than that of ACh,gt, whereas the enthalpy of
ACh(H2O),tt,qa is 10.9 kJ/mol (2.6 kcal/mol) less stable than
that of ACh(H2O),gt,qa. The difference in stability is 11.3 kJ/
mol (2.7 kcal/mol) for ACh(C6H6),gt,qa and ACh(C6H6),tt,qa.
Similar data are obtained for the other rotamers. This comparison
indicates that if internal solvation affects the external solvation
in these complexes, the effect is certainly small, in agreement
with the above conclusion from the experimental data.
I. AChE-ACh Interactions. Acetylcholinesterase (AChE)
is a serine esterase that hydrolyzes ACh at a rate close to the
diffusion-controlled limit.51,52 The three-dimensional structures
of Torpedo californica AChE and a complex between Torpedo
californica AChE and the transition state analogue inhibitor
m-(N,N,N-trimethylammonio)-2,2,2-trifluoroacetophenone (TMT-
FA) have recently been solved by X-ray analysis.1,3 The
crystallographic data combined with data from site-directed
mutagenesis,53-55 kinetic and spectroscopic,52,56,57 chemical
modification,58 and molecular modeling1,55,59,60 studies have
provided important insights into the high specific activity of
AChE. One of the most intriguing features of the AChE structure
is a narrow cavity, about 20 Å long, that penetrates nearly to
the center of the enzyme and contains the active site.1,3 This
cavity, designated the “aromatic gorge”, is lined with 14 highly
conserved aromatic amino acids.61
catalysis occur. The oxyanion of the tetrahedral intermediate
interacts with the NH groups of the residues constituting the
oxyanion hole, namely Gly-118, Gly-119, and Ala-201.
The remaining three subsites are highly aromatic in amino
acid content. The trimethylammonium group of the substrate
fits snugly in a concave binding site formed by the Trp-84, Glu-
199, and Phe-330 residues and three water molecules in the
(misnamed) anionic subsite. Cationic ligands are selectively
bound to this site. The acyl pocket comprises residues Gly-
119, Trp-233, Phe-288, Phe-290, and Phe-331 and provides a
tight fit for the acetoxy methyl group, thereby excluding ligands
of larger size. Finally, the peripheral subsite is located at the
opening of the aromatic gorge and residues Trp-279, Tyr-70,
and Asp-72 are key components of this region. It has been
proposed that the peripheral binding site serves to increase the
concentration of ACh at the top of the cavity and, together with
other aromatic amino acids lining the gorge, to provide an array
of low-affinity binding sites to facilitate the passage of ACh to
the active site.1,63-65 Another function of the aromatic residues
may be to assist in desolvating ACh.7
The roles suggested above for the aromatic residues are
supported by the relative binding energies found in our work.
The binding energies of ACh and the model ion (CH3)4N+ to
aromatic molecules are similar to those to a H2O molecule, but
weaker than those to polar organic molecules (Table 4).
Although the measurements extend only to one or two solvent
molecules and do not account for bulk effects, they suggest that
aromatic residues can supply effective stabilization that allows
entry from the aqueous solution yet avoids trapping of ACh by
polar oxygen or amide groups of the protein. Thus, the
measurements provide a quantitative basis for the proposed
substrate binding to low-affinity sites followed by diffusion to
the active site.1,63-65
(i) Selectivity. The present results show that bond strengths
of aromatic rings to quaternary ions, 33-42 kJ/mol (8-10 kcal/
mol), are much weaker than those to Na+, 117.2 kJ/mol (28.0
kcal/mol),66 or to K+, 80.3 kJ/mol (19.2 kcal/mol).67 Selective
binding to quaternary ions therefore requires other factors.
Geometric constraints may supply one such factor, as the
separation of aromatic residues in the wide channel1-3 may
allow effective multiple interactions with the large quaternary
groups, but not with alkali metal ions.
The active site of AChE contains two primary subsites, the
esteratic and anionic subsites.1,3,56 Two secondary subsites, the
peripheral subsite and the acyl pocket, also have important roles
in the catalytic mechanism.1,3,62 The esteratic subsite lies about
4 Å from the bottom of the cleft, contains the catalytic triad of
Ser-200 (torpedo sequence numbering), His-440, and Glu-327,
and is the site at which the acylation and deacylation steps of
The selectivity may be enhanced by the ACh ion itself through
a conformational change as the ACh leaves the aqueous
environment. A conformational change may contribute to
selectivity for ACh as it is of course not possible for alkali metal
ions or for rigid quaternary ions. Our limited model suggests a
number of ways in which a change in conformation and
improved internal solvation can help stabilize ACh as it goes
from the aqueous to the aromatic environment (Table 5). For
example, assuming ACh is in the gt conformation in solution,
if it desolvates to gt and rearranges to gg′, an estimated 5 kJ/
mol (1.2 kcal/mol) in enthalpy and 1.5 kJ/mol (0.4 kcal/mol)
in free energy could be gained. If it rearranges to gg, an
estimated 3 kJ/mol (0.7 kcal/mol) in both enthalpy and free
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