The contribution of the oxyanion
hole to the functional architecture and to the hydrolytic efficiency of
human acetylcholinesterase (HuAChE) was investigated through single
replacements of its elements, residues Gly-121, Gly-122 and the
adjacent residue Gly-120, by alanine. All three substitutions resulted
in about 100-fold decrease of the bimolecular rate constants for
hydrolysis of acetylthiocholine; however, whereas replacements of
Gly-120 and Gly-121 affected only the turnover number, mutation of
residue Gly-122 had an effect also on the Michaelis constant. The
differential behavior of the G121A and G122A enzymes was manifested
also toward the transition state analog
m-(N,N,N-trimethylammonio)trifluoroacetophenone
(TMTFA), organophosphorous inhibitors, carbamates, and toward selected noncovalent active center ligands. Reactivity of both mutants toward
TMTFA was 2000-11,000-fold lower than that of the wild type HuAChE;
however, the G121A enzyme exhibited a rapid inhibition pattern, as
opposed to the slow binding kinetics shown by the G122A enzyme. For
both phosphates (diethyl phosphorofluoridate, diisopropyl
phosphorofluoridate, and paraoxon) and phosphonates (sarin and soman),
the decrease in inhibitory activity toward the G121A enzyme was very
substantial (2000-6700-fold), irrespective of size of the alkoxy
substituents on the phosphorus atom. On the other hand, for the G122A
HuAChE the relative decline in reactivity toward phosphonates
(500-460-fold) differed from that toward the phosphates (12-95-fold).
Although formation of Michaelis complexes with substrates does not seem
to involve significant interaction with the oxyanion hole, interactions
with this motif are a major stabilizing element in accommodation of
covalent inhibitors like organophosphates or carbamates. These
observations and molecular modeling suggest that replacements of
residues Gly-120 or Gly-121 by alanine alter the structure of the
oxyanion hole motif, abolishing the H-bonding capacity of residue at
position 121. These mutations weaken the interaction between HuAChE and
the various ligands by 2.7-5.0 kcal/mol. In contrast, variations in
reactivity due to replacement of residue Gly-122 seem to result from
steric hindrance at the active center acyl pocket.
 |
INTRODUCTION |
The catalytic efficiency of acetylcholinesterase
(AChE,1 EC 3.1.1.7) and its
high reactivity toward a variety of covalent and noncovalent inhibitors
seem to originate from the unique architecture of the active center,
currently investigated by x-ray crystallography (1-4) and
site-directed mutagenesis (5-10). The x-ray structures of AChE are
characterized by a deep and narrow "gorge," which penetrates
halfway into the enzyme and contains the catalytic site at about 4 Å from its base (1). Several functional subsites in the active center
gorge were identified, including the catalytic triad
(Ser-203(200),2
His-447(440), and Glu-334(327)) (1, 5, 11, 12),
the acyl pocket (Phe-295 (288) and Phe-297(290))
(6, 7, 9), and the "hydrophobic subsite." The latter accommodates
the alcohol portion of the covalent adduct (tetrahedral intermediate)
and may include residues Trp-86(84),
Tyr-133(130), Tyr-337(330), and Phe-338(331), which operate through nonpolar and/or stacking
interactions, depending on the substrate (6, 10, 13). Stabilization of the charged moieties of substrates and other ligands at the active center is mediated by cation-
interactions with the residue at position 86 rather than through true ionic interactions (1, 2, 6, 12,
13). Another important component of the AChE active center functional
architecture is an arrangement of hydrogen bond donors that can
stabilize the tetrahedral transition enzyme-substrate complex through
accommodation of the negatively charged carbonyl oxygen (14).
Structural and modeling studies (1, 6, 15) and, in particular, the
recent solution of the x-ray structure of the transition state analog
TMTFA complexed with TcAChE (3) revealed a three-pronged oxyanion hole
formed by peptidic NH groups Gly-121(118),
Gly-122(119), and Ala-204(201), in contrast to
the two-pronged oxyanion holes in most of serine and cysteine proteases (16). The contribution of the amide nitrogen of Ala-204, rather than
that of the catalytic Ser-203, to the oxyanion hole is consistent with
the reverse handedness of the catalytic triads in AChE compared with
serine proteases (1). Residues Gly-121, Gly-122, and Gly-120 are part
of a flexible "glycine loop" which constitutes one of the gorge
walls adjacent to the catalytic serine and, due to the narrow
dimensions of the gorge bottom, should be in contact with most of the
AChE noncovalent ligands (1, 2, 4). The notion that, apart from its
role in accommodating the oxyanion, this loop is one of the important
determinants of the active center geometry was recently supported by
the x-ray structure of the huperzine A-TcAChE complex (4). In this
structure the conformation of Gly-121 is different from that observed
in other TcAChE-ligand complexes, demonstrating the flexibility of the
loop and the extent of its interaction with the ligand. Such
conformational mobility of the glycine loop implies that its function
may be modified by single replacements of Gly-120(117),
Gly-121(118), and Gly-122(119). It was reported
(17, 18) that replacements of some of the analogous glycine residues in
butyrylcholinesterase resulted in enzyme (G115(117)A)
exhibiting diminished affinity toward tacrine, but not toward BW284C51,
or in enzymes (G117(119)E and the G117(119)H) retaining a nearly wild type catalytic activity toward
butyrylthiocholine (BTC). In HuAChE, our past attempts to introduce
large residues (e.g. histidine, glutamate, and serine) at
position 122 yielded no protein, underscoring the different active
center void volumes of the two enzymes.
Here we describe the construction of HuAChE enzymes with modified
oxyanion hole, through substitution of Gly-121 and Gly-122 by alanine,
and examine their reactivity toward substrates and a variety of
covalent and noncovalent active center inhibitors. We show that
reactivity of the mutants is indeed affected mainly by steric and
conformational changes in the glycine loop. Furthermore, the similar
changes in catalytic activity due to replacement of the adjacent
residue Gly-120, which is not part of the oxyanion hole, may be also
attributed to conformational mobility of the glycine loop. These
results further define the functional architecture of HuAChE-active
center and its role in the enzyme reactivity.
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EXPERIMENTAL PROCEDURES |
Mutagenesis of Recombinant HuAChE and Production of
Mutants--
Mutagenesis of AChE was performed by DNA cassette
replacement into a HuAChE sequence variant (Ew4) which conserves the
wild type (22) coding specificity but carries new unique restriction sites (5). Substitution of residues G120A and G121A and G122A was
performed by replacement of an EspI-NarI DNA
fragment with synthetic DNA duplexes carrying codon GCC(Ala) at the
corresponding mutated positions. All the synthetic DNA
oligodeoxynucleotides were prepared using the automatic Applied
Biosystems DNA synthesizer. The sequences of all new clones were
verified by the dideoxy sequencing method (U. S. Biochemical Corp.
Sequenase kit). The recombinant HuAChE mutants were expressed in
tripartite vectors which allow expression of the cat
reporter gene and the neo selection marker (5, 23).
Recombinant HuAChE and its mutants were expressed in HEK 293 cells as
described previously (24) using stable recombinant cell clones
expressing high levels of each of the mutants (23).
AChE Ligands--
Structures of the various AChE ligands are
shown in Fig. 1. Acetylthiocholine iodide (ATC),
ethyl(m-hydroxyphenyl)dimethylammonium chloride
(edrophonium),
di(p-allyl-N-methylaminophenyl)pentane-3-one (BW284C51), diisopropyl phosphorofluoridate (DFP),
p-nitrophenyl diethylphosphate (paraoxon), physostygmine,
and pyridostigmine were purchased from Sigma.
S-3,3-Dimethylbutyl thioacetate (TB) was synthesized as
described previously (6). Diethyl phosphorofluoridate (DEFP) was
prepared according to the procedure by Saunders and Stacy (19).
Preparation of 2-propyl methylphosphonofluoridate (sarin) and
1,2,2-trimethylpropyl methylphosphonofluoridate (soman) followed an
accepted synthetic procedure using methylphosphonodifluoride (20) and
the appropriate alcohol.
m-(N,N,N-Trimethylammonio)trifluoroacetophenone (TMTFA) was prepared according to the procedure described by Nair et al. (21).
Kinetic Studies and Analysis of Data--
AChE activity was
assayed according to Ellman et al. (25) (in the presence of
0.1 mg/ml bovine serum albumin, 0.3 mM
5,5'-dithiobis(2-nitrobenzoic acid), 50 mM sodium-phosphate
buffer, pH 8.0, and various concentrations of ATC), carried out at
27 °C, and monitored by a Thermomax microplate reader (Molecular
Devices).
Values of inhibition constants (Ki) for the
noncovalent inhibitors edrophonium, tacrine, and huperzine A were
determined from the effects of various concentrations of the inhibitor
on Km and Vmax of the
enzyme-catalyzed hydrolysis of ATC. All the HuAChE enzymes examined
formed rapid equilibria with the tested inhibitors, allowing for an
immediate addition of increasing amounts of enzyme to the ATC/inhibitor
mixture (preincubation of the enzymes with huperzine A for 10 min,
before addition of the substrate, or simultaneous mixing yielded the
same results). The values of Ki were computed
from the secondary plots of the values of
Km/Vmax (determined from
slopes of 1/V versus 1/[S]) versus
concentrations of the respective inhibitors as described previously
(26).
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(Scheme 1)
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The rate constants of progression of the carbamylation reactions
(see Scheme 1) were estimated for at least four different concentrations (and at least 10-fold in ligand concentration, around
the estimated value of Kd) of carbamate (CX), by
adding substrate at various time intervals, and measuring the enzyme
residual activity (E). The apparent bimolecular
carbamylation rate constants (ki), at different
carbamate concentrations, were computed from the plot of slopes of
ln(E) versus time. Only the initial slopes were
considered in order to minimize the errors due to reactivation of the
carbamylated enzymes. Double-reciprocal plot of ki
versus [CX] were used to compute k2
and Kd from the intercept and from the ratio of the
slope and the intercept, respectively, according to the following
equations: 1/k = 1/k2 + 1/ki[CX]; Kd = k2/ki (27). Note that when
k
1
k2,
Kd approaches the value of dissociation constant for
the corresponding Michaelis complex.
Determination of the apparent bimolecular rate constants
(ki) for the irreversible inhibition of HuAChE
enzymes by organophosphates and organophosphonates as well as
estimation of the dissociation constants Kd and the
first-order phosphorylation rate constants (k2)
for paraoxon and DFP were carried out as described before (10, 13).
The apparent first-order rate constants for the
time-dependent inhibition of the wild type and the G122A
HuAChE enzymes by TMTFA were determined by periodically measuring the
initial rate of substrate hydrolysis of aliquots of the reaction
mixture. The inhibitor concentrations used were 7.5-75 nM
for the wild type and 5.0-50 µM for the G122A HuAChEs.
Following the kinetic treatment of Nair et al. (21) and
assuming a two-state inhibition mechanism (Scheme 2), the values of
kon and koff could be
estimated from the linear plots of kobs
versus inhibitor concentration according to Equation 1.
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(Eq. 1)
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(Scheme 2)
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Since in aqueous solution TMTFA is a mixture of the free ketone
(TMTFAket) and the ketone hydrate (TMTFAhyd),
corrected values of the association rate constants were obtained
from kon = k'on (1 + [TMTFAhyd]/[TMTFAket]), using the
ratio of hydrated and ketone forms of TMTFA (62,500), as determined by
19F NMR (21).
To measure directly the values of koff, the
enzyme was inhibited by excess TMTFA (over 90% inhibition), and the
mixture was filtered rapidly through a column (Ultrafree-Biomax30k,
Millipore) to remove free inhibitor. Regeneration of enzymatic activity
for the wild type and the G122A enzymes followed first-order kinetics, yielding values of the dissociation rate constants
(koff). For adduct of the G121A HuAChE, activity
was completely restored within processing time required for removal of
the free inhibitor. TMTFA behaved toward this enzyme as a rapid
reversible inhibitor, and the corresponding inhibition constant
(Ki) could be determined as described above for
noncovalent inhibitors.
Molecular Modeling--
Models of the tetrahedral adducts of
wild type, G120A, G121A, and G122A HuAChEs with ATC and TMTFA were
performed on an Indigo 2 workstation using SYBYL modeling software
(Tripos Inc.). Initial models of the substrate adducts were constructed
as described before (6), and those of TMTFA were built in analogy to
the x-ray structure of the TcAChE-TMTFA conjugate (3). The resulting structures were optimized by molecular mechanics using the AMBER and
the MAXMIN force fields (with AMBER charge parameters for the enzyme).
For most of the starting geometries, for adducts of the G120A and G121A
enzymes, a conformational flip of residue at position 121 occurred
during the optimization process.
 |
RESULTS |
Modification of HuAChE Hydrolytic Activity--
Replacement of
residue Gly-121 by alanine resulted in an enzyme with a 100-fold lower
value of the turnover number (kcat) for both ATC
and its noncharged analog TB (Table I;
for structures see Fig. 1), as compared
with the wild type HuAChE. On the other hand, this substitution had
only a limited effect on the Km values for both
substrates. Past studies with these isosteric substrates demonstrated
that in their respective Michaelis complexes with HuAChE the alkoxy
substituents are accommodated in a different manner (6). Thus, the lack
of significant effect on the values of Km for either
substrate may suggest that during the formation of Michaelis complexes
there is no significant stabilization due to interaction of the
substrate carbonyl moieties with the oxyanion hole. Replacement of the
second oxyanion hole element Gly-122 by alanine also resulted in
similar decreases in the values of kcat for ATC
and TB (18- and 15-fold, respectively). However, for this enzyme the
Km value for ATC was also affected (6-fold), whereas
practically no effect was observed on the corresponding value for TB
(Table I). As in the case of the G121A enzyme the nearly equivalent
decrease in the turnover numbers for both substrates indicates that the
structural modification of the oxyanion hole affects mainly
interactions with the substrate acyl moiety. Replacement of Gly-120 by
alanine was carried out assuming that although this residue is not a
constituent of the oxyanion hole, its substitution may affect the
conformation of the glycine loop. Indeed, the effects on the catalytic
parameters for ATC are similar to those observed for the G121A enzyme
(see Table I), suggesting that the structure of the loop may be
similarly affected by the two replacements. However, since the poorly
expressed G120A enzyme could be obtained only in extremely low
quantities and due to the limited solubility of TB in water, kinetic
studies with the noncharged substrate could not be carried out.
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Table I
Kinetic constants for ATC and TB hydrolysis by HuAChE and its
derivatives
Values represent mean of triplicate determinations with standard
deviation not exceeding 20%.
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Fig. 1.
Chemical formulas of AChE substrates and
inhibitors used in this study. A, substrates and transition
state analog; B, carbamates; C, phosphates;
D, phosphonates; E, noncovalent inhibitors
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Reactivity toward the Transition State Analog TMTFA--
The
kinetics of AChE inhibition by TMTFA has been studied extensively since
the tetrahedral covalent adduct is believed to mimic the transition
state of the acylation process (21, 28, 29). TMTFA was shown to behave
as a tight binding time-dependent inhibitor, a behavior
characteristic of other trifluoroketone inhibitors of serine proteases
(30, 31). In the recently published x-ray structure of TcAChE-TMTFA
adduct (3), the bound ligand is thought to provide a constrained analog
of ACh tetrahedral intermediate, with the oxyanion projecting toward
the NH functions of the oxyanion hole. Therefore, reactivity of TMTFA
toward enzymes carrying replacements of the oxyanion hole elements
Gly-121 and Gly-122 may provide a sensitive measure of the resulting
structural changes in the HuAChE active center.
As for the wild type HuAChE, TMTFA is a time-dependent
inhibitor of the G122A enzyme, showing a linear dependence of the
pseudo first-order rate constants of inhibition
(kobs) on inhibitor concentrations (Fig.
2B). The bimolecular rate
constants kon and the dissociation rate
constants koff were calculated from the relation
kobs = kon [TMTFA]
+ koff and corrected for hydration of the free
ketone (see "Experimental Procedures"). Values of
koff for the wild type and the G122A enzymes
were also determined directly by monitoring the regeneration of
hydrolytic activity from the corresponding adducts and found to be in
good agreement with those determined according to Equation 1 (Table
II). The value of
kon for the G122A enzyme is 300-fold lower than
that for the wild type HuAChE (Table II), indicating that formation of
the tetrahedral adduct may be hindered by some effect related to the
replacement of Gly-122. This is consistent with the observation that
the resulting HuAChE G122A-TMTFA adduct dissociates at about the same
rate as that of the wild type enzyme complex (Table II) and therefore
may be stable enough to result in the observed overall kinetic behavior of time-dependent inhibition. Since with regard to oxyanion
accommodation formation of the tetrahedral adduct with TMTFA should
resemble the formation of analogous adduct with substrate like ATC or
TB, and the effect of Gly-122 substitution on the value of
kon should be comparable to the corresponding
values of kapp. The data in Tables I and II show
that this is indeed the case for ATC which like TMTFA requires
accommodation of the cationic trimethylammonium group by the anionic
subsite Trp-86.

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Fig. 2.
Inhibition of the wild type, G121A, and G122A
HuAChEs by the transition state analog TMTFA. A, example of
inhibition time course of wild type HuAChE ( ) by 7.5 nM
TMTFA and of G121A HuAChE ( ) and G122A HuAChE ( ) in the presence
of 5.0 µM TMTFA. Note that the inhibition progression
curve for the G121A enzyme indicates that steady state is formed
rapidly (few minutes). B, dependence of observed first-order
inhibition rate constants (kobs) on TMTFA
concentration. The span of inhibitor concentrations for the wild type
HuAChE (left panel) was 5.0-50 nM and for the
G122A enzyme (right panel) 0.5-5.0 µM. The
rate constants kon and
koff were calculated from the slope and
intercept, respectively, of these linear fits (see Table II).
C, Lineweaver-Burk plots for the G121A enzyme in the absence
( ) and in the presence of TMTFA 6 × 10 8 ( );
3 × 10 7 ( ); 4 × 10 7 ( );
5 × 10 7 ( ); and 7.5 × 10 7
( ). D, the value of TMTFA inhibition constant
Ki for the G121A enzyme was obtained from secondary
plot of the values of Km/Vmax
versus the corresponding concentrations of the
inhibitor (see "Experimental Procedures").
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Unlike the cases of the wild type and G122A HuAChEs, TMTFA was a rapid
reversible inhibitor of the G121A enzyme (see Fig. 2, A,
C, and D). The fast formation of steady state for
the HuAChE G121A-TMTFA adduct, which is characteristic to HuAChE
complexes with noncovalent inhibitors, suggested that the corresponding dissociation is much faster and that TMTFA no longer behaves as a tight
binding inhibitor (32). Indeed, regeneration of the G121A HuAChE
hydrolytic activity, following removal of excess inhibitor, was too
fast to be determined under conditions of the present study, and
therefore only a lower limit of this rate constant could be estimated
(koff
0.15 min
1 as compared
with 1.2 × 10
3 min
1 in the G122A
enzyme; see Table II). This finding suggests that the interactions
contributing to the stabilization of TMTFA in the covalent adduct are
stronger in the cases of the wild type and G122A HuAChE than in case of
the G121A enzyme. Moreover, although the relative destabilization of
the tetrahedral TMTFA adducts with the G122A and G121A enzymes is
similar (4.0 and 5.0 kcal/mol, respectively, see Table II), the
differential effects of the two mutations on the values of the rate
constants and in particular on those of koff
suggest that the destabilizing effects in each case are due to
different phenomena.
Reactivity toward Carbamates and Organophosphorous
Inhibitors--
Carbamate and organophosphate AChE inhibitors react
with the enzyme yielding either slowly decomposing or practically
stable covalent adducts, and therefore their overall inhibitory
potencies are most conveniently measured by the bimolecular rate
constants Ki (see Scheme 1). Although carbamylation
and phosphorylation of the catalytic Ser-203 are quite different
reactions, polarization of the respective C=O and the P=O bonds through
interactions with the oxyanion hole may be essential in facilitating
the nucleophilic addition. On the other hand, the anticholinesterase
potencies of carbamates and organophosphates were shown in the past to
depend mainly upon their complementarity with the active center.
Numerous studies of structure-activity relationships for the various
inhibitors (33-37), as well as for AChE enzymes modified by
mutagenesis (10), suggested that structural variability affects the
affinity toward the enzyme (Kd), rather than the
rate of the covalent adduct formation (k2). In
this context it was quite interesting to examine the effects of
structural modifications in the oxyanion hole on the inhibition
kinetics of HuAChE enzymes by carbamate and organophosphate
inhibitors.
Kinetic studies were carried under conditions that allowed for
derivation of values for Kd and
k2 (see "Experimental Procedures" and Scheme
1) for the prototypical AChE carbamate inhibitors physostigmine and
pyridostigmine. For the wild type HuAChE, the values of
Kd and k2 (Table
III) obtained for physostigmine are
similar to those measured recently in a comprehensive kinetic study of
its reaction with electric eel AChE (37). The bimolecular carbamylation
rate constants of the G122A enzyme by physostigmine and pyridostigmine
decreased to about the same extent relative to the wild type HuAChE
(85- and 56-fold, respectively). For both inhibitors, these changes in
the values of ki were caused by comparable changes
in the respective values of Kd and
k2 (Table III). A somewhat larger decrease in
the bimolecular carbamylation rate constant by physostigmine was
observed for the G121A HuAChE (290-fold relative to the wild type
enzyme). Unlike the case of the G122A enzyme, this diminished value of ki is mainly due to loss of affinity toward the
inhibitor (the corresponding value of Kd was 65-fold
higher than that for the wild type HuAChE). The effect of residue
replacement at position 121 on the affinity toward carbamates may be
even more dramatically demonstrated for pyridostigmine, since in this case kinetic analysis indicated a rapid reversible inhibition (see Fig.
3A). According to the kinetic
model of carbamylation (see Scheme 1), this observation may suggest
that the G121A-pyridostigmine Michaelis complex is destabilized to the
point where the ratio k
1/k2 precludes
observation of the covalent adduct, within the time frame of the
kinetic experiment.
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Table III
Kinetic constants for carbamylation reactions of HuAChE and its
derivatives
Rate and dissociation constants according to Scheme 1 (see
"Experimental Procedures").
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Fig. 3.
Inhibition of the wild type, G121A, and G122A
HuAChEs by pyridostigmine. A, example of inhibition time
course of wild type HuAChE ( ) in the presence of 75 nM
pyridostigmine, of the G122A HuAChE ( ) in the presence of 25.0 µM pyridostigmine, and of the G121A HuAChE ( ) in the
presence of 25 mM inhibitor. B,
double-reciprocal plots of kobs
versus concentrations of pyridostigmine: left
panel, wild type HuAChE (inhibitor concentration range 0.075-0.75
µM); right panel, G122A enzyme (inhibitor
concentration range 10-100 µM).
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Inhibition of the oxyanion hole mutants G122A and G121A by
organophosphorous inhibitors was investigated using the phosphates DFP,
DEFP, and paraoxon as well as the phosphonates soman and sarin.
Comparison of the inhibition rate constants (ki) for
the two mutant enzymes clearly demonstrates the different effects of
the two replacements on enzyme reactivity toward these inhibitors. Upon
replacement of Gly-121 by alanine the reactivity toward all the
inhibitors decreased by more than 3 orders of magnitude (see Table
IV). On the other hand, for the G122A
HuAChE the inhibition rate constants of phosphates decrease
10-100-fold, relative to the wild type enzyme, whereas those of
phosphonates are more affected (460-500). Determination of the values
of Kd and k2 for paraoxon and
DFP inhibition of the G122A and the G121A HuAChEs has indicated that
for the latter enzyme affinity toward the inhibitors rather than the
actual rate of the phosphorylation step (k2) is mainly affected (Table V).
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Table IV
Bimolecular rate constants for phosphylation reactions of HuAChE and
its oxyanion hole mutants
The term phosphylation refers to all the nucleophilic reactions at
tetracoordinated phosphorus atom (56).
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Table V
Kinetic constants for phosphorylation reactions of HuAChE and its
derivatives
Rate and dissociation constant were determined as described before
(10).
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Reactivity toward Noncovalent Active Center Inhibitors--
As
already mentioned, the glycine loop is also a structural element of the
active center and therefore may be part of the binding environment for
ligands which do not contain the oxyanion moiety. Moreover, its
capacity to participate in stabilization of noncovalent complexes is
limited due to the absence of side chains. To examine the effect of
modified loop structure on stabilization of noncovalent complexes, the
inhibitory activities of two active center ligands edrophonium and
huperzine A as well as that of the bisquaternary ligand BW284C51,
toward the G121A and G122A HuAChEs, have been evaluated.
Replacement of either Gly-121 or Gly-122 by alanine had only a minor
effect on the inhibitory activity of the bisquaternary ligand BW284C51,
showing that the structure of the active center gorge is essentially
unchanged (Table VI). For inhibition of
the G122A enzyme by edrophonium and more so for inhibition of both mutant enzymes by huperzine A, pronounced effects were observed relative to the wild type HuAChE (Table VI). Since these ligands do not
contain groups capable of interacting with the oxyanion hole, the
reason for the diminished inhibitory activity is probably steric
obstruction due to the modified structure of the glycine loop. For
huperzine A such a conclusion is consistent with the recently published
x-ray structure of the TcAChE-huperzine A complex where the ligand is
tightly fitted against the glycine loop, altering its conformation
relative to structures of other noncovalent TcAChE complexes (4).
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Table VI
Competitive inhibition constants of HuAChE and its derivatives by
active center ligands
Values represent mean of triplicate determinations with standard
deviation not exceeding 20%. The correlation coefficients of the
linear plots were at least 0.95.
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 |
DISCUSSION |
The contribution of oxyanion hole to AChE catalytic efficiency was
postulated on the basis of analogy to other serine hydrolases and on
theoretical studies (38-40). For these hydrolases, and in particular
for serine proteases, such structural element has been identified in
x-ray structures of covalent adducts that mimic the corresponding
tetrahedral intermediates in reactions with substrates (14, 16). In
proteases of the chymotrypsin family, the oxyanion hole consists of the
backbone NH groups of Gly-193 and Ser-195, whereas in subtilisins one
of the H bond donors is the side chain amine group of Asn-155. The
three-pronged oxyanion hole identified in the x-ray structure of TcAChE
(1, 3) is similar to the corresponding functional motifs observed in the structures of Candida rugosa and Rhizomucor
miehei lipases (41). These lipases share with AChE the
/
-hydrolase fold (42); however, in the cases of AChE and CRL the
oxyanion hole is preformed and does not change significantly upon
inhibitor binding (43), whereas that of RML is formed only upon
enzyme-ligand complex formation (44).
Despite the importance of these studies in providing structural
evidence for the existence of an oxyanion hole, it still remains unclear how interactions of this motif with substrate contributes to
catalytic efficiency (16). Henderson (45) suggested a specific binding
site for the oxyanion that lowers the free energy of the tetrahedral
intermediate favoring its formation. It was argued that stabilization
of the oxyanion by pre-aligned dipoles of the oxyanion hole is more
effective than in water, as solvent reorganization is avoided (46).
More recently, the NH groups of the oxyanion hole were proposed to
participate in a "concerted general acid-general base-catalyzed
formation of a tetrahedral intermediate" (47). The partial transfer
of protons to the forming oxyanion results in short, strong hydrogen
bonds which may account for much of the enzymatic rate
acceleration.
A direct and quantitative evaluation of the oxyanion hole role in
catalysis was carried out for enzymes, such as subtilisin or papain,
where one of the hydrogen bonding groups was on a side chain and could
be removed through residue replacement (16). For subtilisin, several
replacements of residue Asn-155 resulted in significant decreases of
the kcat/Km values (48-50), corresponding to a change in transition state stabilization in the
range of 3-5 kcal/mol. Most of the effect could be attributed to a
decrease in values of kcat, suggesting that
interactions with the oxyanion hole do not participate in stabilization
of the Michaelis complex (16). For subtilisin and for serine proteases in general, such an outcome may not be surprising since the complexes are thought to involve numerous polar and hydrophobic interactions between the enzymes and their bulky peptidic substrates (14, 51).
However, comparison of these enzymes with AChE raises the question
whether the initial accommodation of small substrates and covalent
inhibitors by AChE follows the same pattern with regard to oxyanion
hole participation.
Functional Role of Residue Gly-121 in Oxyanion Hole--
The G121A
HuAChE enzyme exhibited impaired catalytic activity toward ATC and TB
mainly due to decrease in the respective values of
kcat. Thus, introduction of small side chain
onto the HuAChE oxyanion hole residue does not appear to interfere in
the step of Michaelis complex formation. Since only limited atomic
motion is thought to take place during the transition from planar to tetrahedral substrate geometries (46), this side chain probably does
not interfere with formation of the transition state. Therefore, the
lower values of the turnover number for the G121A enzyme could be a
consequence of relative destabilization of the tetrahedral intermediates due to impaired binding capacity of the oxyanion hole.
Moreover, the extent of this destabilization (2.7-3.4 kcal/mol; monitored by the rate constant of the acyl-enzyme formation
kapp; see Table I) is in good agreement with
that mentioned above for subtilisins mutated at position 155 (49),
implying that part of the H-bonding capability of the oxyanion hole in
the HuAChE mutant enzyme is lost. The notion that replacement of
Gly-121 by alanine changes the structure of the oxyanion hole in HuAChE and consequently affects its H-bonding with the tetrahedral transition state is consistent with the effect of substituting the adjacent residue Gly-120. Although residue Gly-120 is not part of the oxyanion hole, its replacement by alanine had nearly equivalent effect to that
of replacing Gly-121, on the kinetic profile of ATC hydrolysis. Since
residues Gly-120 and Gly-121 are part of a conformationally mobile
glycine loop, the most straightforward rationalization of this
similarity is that replacement of either Gly-120 or Gly-121 may bring
about the same structural change of the oxyanion hole. The recently
reported replacements of residue Gly-115 in butyrylcholinesterase (equivalent to Gly-120 in HuAChE) by alanine and by serine also affected mainly the respective values of kcat
(18).
The effect of mutation at position 121 on reactivity toward the
transition state analog TMTFA is also consistent with the idea of a
partial loss of the oxyanion H-bonding capacity. The inhibition
characteristics of the ligand was changed from a slow inhibitor of the
wild type HuAChE, into a rapidly equilibrating one for the G121A enzyme
(Table II). The main observable difference in kinetic behavior,
relative to the adduct of the wild type enzyme, was a significant
increase in the dissociation rate (koff),
indicating a lower stability of the G121A-TMTFA covalent conjugate.
Since the tripartite stabilization due to the oxyanion hole (Gly-121, Gly-122, and Ala-204) is a major component of the TMTFA tetrahedral adduct accommodation (3), it is reasonable to assume that such destabilization may result from partial destruction of the oxyanion hole motif.
The possible functional impairment of the oxyanion hole, in the G120A
and G121A enzymes, was also assessed by molecular modeling of the
corresponding tetrahedral intermediates with ATC. It appears that
introduction of methyl groups results in both cases in a conformational
flip of residue at position 121, removing the H-bond donating NH moiety
from the vicinity of the oxyanion (see Fig. 4, B and C). In the
resulting models, the H-bond interactions with residues Gly-122 and
Ala-204, as well as the substrate orientation relative to the active
center binding environment, resemble closely those observed in the
corresponding model of the wild type enzyme (Fig. 4A). The
models suggest also that substitution at positions 120 and 121 by
alanine does not result in steric interference in the HuAChE-ATC
tetrahedral species (Fig. 4). In addition, the proposed orientation of
the Ala-121 methyl group is also consistent with the inhibitory
activities of the noncovalent active center ligands, edrophonium and
BW284C51 toward the G121A enzyme. The minor differences of
Ki values for edrophonium and BW284C51 as compared
with those for the wild type HuAChE seem to be a result of residue
Ala-121 interaction with the tetramethylammonium moiety of edrophonium
and with the aryl ring of BW284C51 (models not shown).

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Fig. 4.
Stereo view of models of ATC tetrahedral
intermediates with the wild type, G120A, and G121A HuAChE
enzymes. H-bond interaction distances between ATC oxyanion and
components of the oxyanion hole are marked by dashed lines.
A, wild type HuAChE-ATC tetrahedral conjugate, the oxyanion
is within 2.9, 2.8, and 2.9 Å from the amide NH moieties of Gly-121,
Gly-122, and Ala-204, respectively. B, G120A-ATC tetrahedral
conjugate, the Ala-120 methyl carbon is shown as a ball, and
the oxyanion is within 2.8 and 3.2 Å from the amide NH moieties of
Gly-122 and Ala-204, respectively. Note that the increased distance of
the NH moiety of Gly-121 from the oxyanion of ATC (3.9 Å) as well as
their relative orientation preclude H-bonding between Gly-121 and ATC.
C, G121A-ATC tetrahedral conjugate, the Ala-121 methyl
carbon is shown as a ball, and the oxyanion is within 2.8 and 3.0 Å from the amide NH moieties of Gly-122 and Ala-204,
respectively. The positioning of the NH moiety of Ala-121 is similar to
that of Gly-121 in B.
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The oxyanion hole is thought to be one of the elements of the AChE
functional architecture, participating in formation of Michaelis
complexes with organophosphorous inhibitors (10, 15, 52). Indeed, the
dissociation constants (Kd) of the G121A enzyme
complexes with paraoxon and DFP, respectively, were 188- and 214-fold
higher than those for the wild type HuAChE (see Table IV). The finding
that the dissociation constants for the two phosphates, differing in
the bulk of their alkoxy substituents (see Fig. 1), show large but
similar variations indicates that the relative destabilization of the
corresponding G121A-Michaelis complexes is not due to steric effects
but rather due to impaired accommodation of the phosphoryl oxygen.
Furthermore, the bimolecular rate constants for inhibition of the G121A
enzyme, by all the phosphates or phosphonates examined
(ki, see Table IV) show similar decreases relative
to the wild type HuAChE. These findings further support the assumption
that the only significant functional change in the G121A enzyme is the
diminishing of H-bonding capacity of the oxyanion hole and its
capability to accommodate the phosphoryl moiety. The overall loss in
interaction energy between the G121A enzyme and the organophosphorous
inhibitors (4.5-5.2 kcal/mol; calculated from values of the ratio
kimutant/kiwild type
shown in Table IV), relative to the wild type HuAChE, is much larger
than the corresponding decrease for substrates, suggesting a somewhat
different role of the oxyanion hole motif in HuAChE phosphorylation and
in acylation by substrates. The reason for such a considerable effect
on the enzyme reactivity toward organophosphorous agents may be that
polarization of the P=O bond during complex formation is necessary for
activating the inhibitor for nucleophilic attack and is facilitated by
its tetrahedral geometry.
In view of the proposed difference in the initial accommodation of
tetrahedral phosphates and planar substrates by the oxyanion hole, it
was interesting to examine the reactivity of the G121A enzyme toward
carbamates. For these inhibitors, the planar geometry characteristic to
substrates is combined with much lower reactivity of the carbamyl
moiety, and thus, activation may be required to assist a nucleophilic
addition. Therefore, as in the case of phosphates, activation may be
achieved by juxtaposing the carbamyl oxygen with the oxyanion hole so
that the C=O bond is strongly polarized already in the Michaelis
complex. Indeed, from the values of kinetic constants
(Kd, k2) for inhibition of
the G121A enzyme by physostigmine, relative to the wild type HuAChE
(see Table III), it is evident that the mutation affects predominantly
the stability of the Michaelis complex. In case of inhibition of the G121A enzyme by pyridostigmine, the dissociation rate of the
destabilized Michaelis complex may become much higher than the rate of
acylation leading to the observed reversible association with this
carbamate.
Functional Role of Residue Gly-122 in Oxyanion Hole--
Residue
Gly-122 is another element of the HuAChE oxyanion hole located on the
flexible glycine loop in the active center. Indication that glycine at
position 122 may not be a prerequisite for a fully functional oxyanion
hole can be inferred from the sequence of certain cholesterol esterases
and lipases, which also belong to the
/
-hydrolase fold (54), and
where the motif Gly-120-Gly-121-Gly-122 is replaced by Gly-Gly-Ala.
Furthermore, in butyrylcholinesterase the somewhat wider base of the
active center gorge (53) allows for substitutions of the analogous
oxyanion hole constituent Gly-117, with only a small effect on the
catalytic activity (17, 18). On the other hand, the catalytic activity
of the both the G121A and the G122A HuAChEs toward ATC was 100-fold
lower than that of the wild type enzyme. However, for the latter the
decrease of the bimolecular rate constant of acylation
(kapp) was due to variation in both the values
of Km and kcat, implying that
substitution of position 122 by alanine affected the function of the
oxyanion hole in a different manner than the corresponding substitution
at position 121.
The difference in reactivity pattern of the G121A and G122A enzymes
toward the transition state analog TMTFA further underscores the
different effects of replacing Gly-121 or Gly-122 on the function of
the oxyanion hole. Unlike the case of the G121A enzyme, the slow
decomposition rate of the TMTFA adducts of both the wild type and G122A
enzymes suggests that for the latter stabilization due to the oxyanion
hole is mostly unaffected. On the other hand, the decrease of the rate
constant of HuAChE G122A-TMTFA adduct formation
(kon) is comparable to that of the G122A enzyme
acylation by ATC (kapp) (decrease of 300- and
100-fold, respectively, compared with the wild type enzyme), suggesting
a similar interference in both processes. A model of the G122A-TMTFA
adduct, constructed on the basis of the x-ray structure of the
corresponding TcAChE conjugate (3), shows that the methyl group of
Ala-122 protrudes into the acyl pocket restricting the space occupied
by the trifluoromethyl group of TMTFA (see Fig.
5). Since the latter is only slightly larger than a methyl group, analogous steric interference can be
expected also for the tetrahedral intermediates of substrates.

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Fig. 5.
Stereo view of models of TMTFA tetrahedral
conjugates with the wild type and G122A HuAChE enzymes. H-bond
interaction distances between the oxyanion and components of the
oxyanion hole are marked by dashed lines. A, wild
type HuAChE-TMTFA conjugate, constructed according to the x-ray
structure of the corresponding adduct of TcAChE (3), where the oxyanion
is within 3.0, 2.9, and 2.7 Å from the amide NH moieties of Gly-121,
Gly-122, and Ala-204, respectively. B, G122A-TMTFA
conjugate. Note that accommodation of the trifluoromethyl group of the
ligand is hindered by interaction with the methyl group of Ala-122 as
demonstrated by partial overlap of their respective volumes (shown as
dotted area). The oxyanion hole is within 2.7 Å from the
amide NH moieties of Gly-121 and Ala-122 (dashed lines), and
within 3.2 Å from the amide nitrogen of Ala-204 (residue not
shown)
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In accordance with the notion that replacement of residue Gly-122 by
alanine introduces steric interference in the acyl pocket of HuAChE, we
find that this substitution affects differently the rate constants of
phosphorylation by phosphates compared with phosphonates (Table III).
For the phosphonates, soman and sarin, the decrease in values of
ki, relative to the wild type enzyme (500- and
460-fold, respectively), was about an order of magnitude larger than
for the phosphates. This reactivity pattern appears to be consistent
with the proposed restriction of space available in the acyl pocket,
since the methyl group introduced to this subsite by phosphonates is
more bulky than the alkoxy oxygen presented by the phosphates. The
localized nature of this steric perturbation is indicated also by the
fact that it similarly affects the values of ki for
phosphonates despite the different sizes of their alkoxy substituents.
For the phosphates DFP and paraoxon most of the effect was observed on
the values of k2 (about 5-fold for both
phosphates), demonstrating that stabilization of the corresponding
Michaelis complexes was relatively unaffected.
The diminished reactivity of the G122A enzyme toward physostigmine and
pyridostigmine can be also accounted for in terms of a steric
constraint in the acyl pocket. Although the size of the molecular
moiety introduced to this site by pyridostigmine (dimethylamino) is
considerably larger than that of methyl group, its trigonal geometry
allows for orientations in which steric conflict with the side chain of
Ala-122 is mostly avoided (model not shown).
Conclusions--
Kinetic study of catalysis and inhibition of the
G120A, G121A, and G122A HuAChE enzymes with variety of covalent and
noncovalent inhibitors suggest that site-directed mutagenesis allows us
to manipulate specifically the structure and interaction
characteristics of an oxyanion hole even though it consists only of
main chain NH groups. Furthermore, from the changes in reactivity of
the mutant enzymes toward the various ligands and, in particular, toward the transition state analog TMTFA, the extent of structural perturbation introduced by these mutations could be assessed. One of
the surprising observations is that formation of HuAChE-substrate Michaelis complexes do not seem to involve substantial interactions with the oxyanion hole. On the other hand, the stability of analogous complexes with covalent inhibitors like organophosphates and carbamates appears to require accommodation by oxyanion hole, probably to activate
the respective phosphoryl or carbamyl groups for nucleophilic addition
of the catalytic Ser-203.