The EMBO Journal vol.11 no.10 pp.3561 -3568, 1992

Substrate inhibition of acetylcholinesterase: residues affecting signal transduction from the surface to the catalytic center Avigdor Shafferman, Baruch Velan, Arie Ordentlich, Chanoch Kronman, Haim Grosfeld, Moshe Leitner, Yehuda Flashner, Sara Cohen, Dov Barak1 and Naomi Ariel Departments of Biochemistry and 'Organic Chemistry, Israel Institute for Biological Research, Ness-Ziona, 70450, Israel Communicated by J.-P.Changeux

Amino acids located within and around the 'active site gorge' of human acetylcholinesterase (AChE) were substituted. Replacement of W86 yielded inactive enzyme molecules, consistent with its proposed involvement in binding of the choline moiety in the active center. A decrease in affmity to propidium and a concomitant loss of substrate inhibition was observed in D74G, D74N, D74K and W286A mutants, supporting the idea that the site for substrate inhibition and the peripheral anionic site overlap. Mutations of amino acids neighboring the active center (E202, Y337 and F338) resulted in a decrease in the catalytic and the apparent bimolecular rate constants. A decrease in affinity to edrophonium was observed in D74, E202, Y337 and to a lesser extent in F338 and Y341 mutants. E202, Y337 and Y341 mutants were not inhibited efficiently by high substrate concentrations. We propose that binding of acetylcholine, on the surface of AChE, may trigger sequence of conformational changes extending from the peripheral anionic site through W286 to D74, at the entrance of the 'gorge', and down to the catalytic center (through Y341 to F338 and Y337). These changes, especially in Y337, could block the entrance/exit of the catalytic center and reduce the catalytic efficiency of AChE. Key words: acetylcholinesterase/peripheral anionic site/ active site

Introduction Acetylcholinesterase (AChE, EC 3.1.1.7) functions at cholinergic synapses by rapid hydrolysis of the neurotransmitter acetylcholine (ACh). AChE catalysis involves an acylation/deacylation step at a serine residue in the active center. Studies of the rate of hydrolysis of various substrates have led to the hypothesis that the active center is composed of two functional subsites: an esteratic subsite and an anionic subsite (for reviews see Rosenberry, 1975; Quinn, 1987). The latter was implicated in orienting the positively charged substrate in the active center. Evidence also exists for an allosteric regulation of AChE activity by ligand binding to an anionic site(s) physically remote from the active site (Changeux, 1966). The role of the peripheral anionic site in AChE function is still unclear. Substrate inhibition of © Oxford University Press

AChE at high ACh concentrations is well documented (Augustinsson, 1948; Quinn, 1987) and it has been proposed that it occurs directly at the active center (Froede et al., 1986) or indirectly through a peripheral site which changes the active center allosterically (Changeux, 1966; Rosenberry, 1975). Recent kinetic studies (Radic et al., 1991) using propidium, a ligand selective for the peripheral anionic site, suggest that the binding site for substrate inhibition and the peripheral anionic site may consist of the same or overlapping structural elements. Energy transfer measurements (Berman et al., 1980) suggest that the distance between the peripheral anionic site and the ative site is 20 A, while studies with bisquatemary ammonium ligands (e.g. decamethonium), which presumably bind to both sites (Krupka, 1966), suggest that the distance is 14 A. In spite of the wealth of information accumulated on AChE, a comprehensive characterization of its structurefunction properties is only recently beginning to emerge. Primary structure comparisons of members of the AChE family (Krejci et al., 1991; Gentry and Doctor, 1991), site directed labeling (Kieffer et al., 1986; Weise et al., 1990; Kreienkamp et al., 1991), mutagenesis studies (Gibney et al., 1990; Neville et al., 1990; Velan et al., 1991b) and resolution of the X-ray structure of Torpedo californica AChE (TcAChE) (Sussman et al., 1991) all provide information pertinent to the understanding of the molecular basis for the catalytic activity. The location and spatial organization of the serine and histidine residues constituting the active site of AChE were determined (Gibney et al., 1990; Shafferman et al., 1992). Mutagenesis of acidic residues that are highly conserved in the ChE superfamily provided biochemical evidence for the involvement of a glutamic residue in the catalytic triad (Shafferman et al., 1992), as also predicted from the X-ray structure of TcAChE (Sussman et al., 1991). These site directed mutagenesis studies revealed also specific aspartic residues involved in salt bridges essential for folding and maintaining the threedimensional (3-D) structure of the enzyme. Site directed labeling studies provided clues for the involvement of various amino acid stretches in ligand binding to anionic sites (Kieffer et al., 1986; Weise et al., 1990). X-ray crystallography also revealed a 20 A deep, narrow 'gorge', that penetrates halfway into the enzyme, containing the catalytic triad 4 A away from its bottom (Sussman et al., 1991). This 'active site gorge' is lined by 14 aromatic amino acids, which may explain results of kinetic studies (Quinn, 1987) and site directed labeling studies that have identified tryptophan (Kreienkamp et al., 1991) and phenylalanine (Kieffer et al., 1986) residues at or near the active center. This complex array of aromatic residues was hypothesized (Sussman et al., 1991) to provide a guidance mechanism facilitating a two-dimensional difusion of ACh to the active site. Yet, these residues can also serve as a relay of molecular events taking place at the peripheral binding sites, presumably located at -

-

3561

A.Shafferman et al.

the surface of the protein close to the rim of the 'gorge'. In order to probe some of these possibilities we have chosen to mutagenize several amino acids within and around the active site 'gorge'. We found that the aromatic and anionic residues in this region affect enzymatic activity at various degrees. Some amino acids play a major role in catalysis, while others could be key elements in the allosteric regulation of AChE activity at high substrate concentrations.

Results Generation of selected AChE mutants The putative spatial locations of the human AChE (HuAChE) residues targeted for mutagenesis were deduced from the X-ray structure of TcAChE (Sussman et al., 1991). HuAChE and TcAChE are likely to have a similar fold as indicated by sequence homology (Schumacher et al., 1986; Soreq et al., 1990) and comparative modeling studies (Barak et al., 1992). All seven amino acids (D74, W86, E202, W286, Y337, F338 and Y341) selected for study appear in and around the active site 'gorge' (Figure 1). The aromatic PAS

Gorge entrance

amino acids of HuAChE W86, Y337 and F338 are analogous to W84, F330 and F331 in TcAChE and electric eel AChE. These amino acids have been shown in separate photoaffinity labeling studies to be part of the catalytic center (Kieffer et al., 1986; Kreienkamp et al., 1991), and the tryptophan residue was actually suggested to be part of the anionic subsite. The HuAChE W86 was therefore replaced with alanine and with the carboxylic glutamic acid; F338 was substituted with alanine and Y337 with alanine and phenylalanine (the latter mimics the TcAChE configuration). D74 is analogous to D70 of butyryl ChE (BChE). We have recently shown (Shafferman et al., 1992) that the HuAChE D74N and D74G mutants can recreate in HuAChE the phenotype of the naturally occurring atypical variant, D70G, of human BChE (McGuire et al., 1989; Neville et al., 1990), and that these replacements do not lead to detectable conformational changes. Since D74 appears to be located close to the entrance of the 'gorge' and since the D74 mutants had altered activity, we generated two additional mutants: one that maintains the negative charge (D74E) and one that carries a positive charge (D74K). Y341, which appears in

PAS

Gorge entrance

Fig. 1. Stereo view of the 'active site gorge' of HuAChE depicting residues mutated in this study. The catalytic triad (S203-H447-E334) is shown in shaded lines. The dimensions of the 'gorge' are demonstrated by a few relevant distances: C-yD74-01S203, 14.7 A; C&yD74-CbE202, 15.8 A; CyD74-centroid of aromatic ring Y337, 8.7 A; obID74-Ot1Y341, 3.6 A; CyD74-NE1W286, 8.4 A; and OyS203-NElW86, 10 A. Double headed arrows indicate the direction of the proposed relay from the peripheral anionic site (PAS) through D74 and down to the active center. to

coio w

0so

00

60

40

:0

do

20 -2 0 0.000

0.015

0.030

[Edrophonium] (mM)

0.045

0.015

0.030

0.045

[Propidium] (mM)

Fig. 2. Inhibition of HuAChE and D74-HuAChE mutants by edrophorium and propidium. Enzyme preparations (5-20 ng/ml in 5 mM buffer phosphate pH 8) were preincubated for 20 min at 27°C with the indicated inhibitors and reaction was initiated by adding ATCh to a final concentration of 0.5 mM. Wild type rHuAChE, open triangle; D74E, closed square; D74N, closed circle; D74G, open circle; D74K, closed triangle.

3562

Relay of substrate inhibition in acetylcholinesterase

the 3-D structure within hydrogen bonding distance from D74, was also targeted for mutagenesis. W286 is C-terminal to a peptide which was shown by site directed labeling studies [peptide 270-277 in TcAChE (Weise et al., 1990)] to form a part of the peripheral anionic site and is located at the entrance of the 'gorge'. Moreover, it was recently shown (Sussman et al., 1992) that the analogous tryptophan residue of TcAChE undergoes a substantial conformational change upon binding of certain AChE inhibitors. Mutations of E199 in TcAChE, N-terminal to the catalytic serine, were reported to yield an enzyme with a higher Km and devoid of substrate inhibition behavior (Gibney et al., 1990). The analogous residue, position 202 of HuAChE, which is located at the bottom of the 'gorge', was replaced with glutamine, aspartic acid and alanine. The secretion of AChE by 293 cells transfected with either wild type or mutagenized HuAChE cDNA was determined. Experiments were performed in quadruplicate and all measurements were normalized to the coexpressed chloramphenicol acetyl transferase (CAT) activity. The AChE protein mass produced in different transfections of any of the 12 different mutants did not vary by more than 2-fold from that obtained from wild type AChE. HuAChE W86 mutants are catalytically defective molecules The 293 cells transfected with plasmids carrying either wild type, W86A or W86E AChE cDNAs secrete similar amounts (80-120 ng/transfection) of AChE polypeptide into the medium. However, none of the W86 mutated molecules exhibited enzyme activity above background. Thus, replacements of tryptophan 86 with alanine or glutamine result in at least a 100-fold reduction in the catalytic potential of the enzyme. The conformational integrity of the W86 mutants was assessed by quantitative ELISAs (Velan et al., 1991b; Shafferman et al., 1992) using four different monoclonal antibodies (AEl, AE2, HR5 and A123). The W86 mutants and the wild type preparations were indistinguishable in these assays (data not shown), suggesting that the loss in catalytic activity in W86A and W86E polypeptides is

probably not a consequence of distorted folding. Except for W86 mutants, all other mutants analyzed in this study exhibited AChE activity. Kinetic studies of active mutants Using the Lineweaver-Burk analysis, the MichaelisMenten constant (K,,,), apparent catalytic first order rate constant (kcat) and apparent bimolecular rate constant (kcatlKm) were calculated for the hydrolysis of acetylthiocholine by the 12 active HuAChE mutants (Table I). In general, the mutated enzymes showed either no change at all in affinity to the substrate or at most a 2- to 4-fold decrease (higher Km values) relative to the wild type AChE. The striking exception was the 35-fold increase in the Km of the D74K mutant. Interestingly, replacements of residues at postion 74 affected the Km more than replacement at any other position, including amino acids vicinal to the active center. On the other hand, kcat was significantly affected only by substitutions at the three amino acids (E202, Y337 and F338) adjacent to the catalytic center, with substitutions at E202 resulting in a decrease of up to 80-fold in the catalytic rate constant as compared with wild type HuAChE. The combined effects on both affinity for substrate (Kin) and kcat in the E202A mutant, resulted in a reduction of > 100-fold in the apparent bimolecular rate constant. These observations are consistent with the unique locations of E202 next to the catalytic S203 and its orientation relative to the catalytic triad histidine 447 (see also below). Inhibition of mutants by edrophonium, propidium, bisquaternary ligands and acetylthiocholine All four D74 mutants are less susceptible to inhibition by edrophonium, a selective active center ligand, than the wild type HuAChE (Figure 2 and Table I). Substitution of aspartate 74 with glutamate had a minimal effect on inhibition by edrophonium, yet substitution with asparagine or glycine had a greater effect than substitution with the positively charged lysine. These results suggest that charge in itself is not the major factor in generating a phenotype with a higher resistance to edrophonium in D74 mutants. The most

Table I. Catalytic functions of HuAChE and its mutants AChE type

Km (mM)

kcat (x i0- X nuin-)

kcat/Km (x 10-8)

(M-1 WT

0.14

0.05

3.7

D74E D74N D74G D74K

0.50 0.58 0.63 4.50

+ 0.15 ± 0.20 ± 0.20 + 0.90

4.4 2.7 3.6 3.5

E202Q E202D E202A

0.35 + 0.01 0.25 + 0.01 0.66 + 0.01

W286A

0.8 1.1 1.0 0.1

8.8 4.6 5.7 0.8

IC50

(mutant)/ICm (WT)a

Edrophonium

Propidium

1

1

2.1 1.3 1.2 1.0

2 7 5 4

0.56 i 0.01 0.15 + 0.03 0.10 ± 0.02

1.6 + 0.6 0.6 + 0.3 0.2 A 0.3

4 22 80

0.28 + 0.06

4.0 ± 0.1

14.3 + 0.5

Y337F Y337A

0.19 + 0.01 0.14 + 0.03

3.7 + 2.0 1.0 + 0.1

19.5 + 1.5 7.1 + 0.3

1 15

F338A

0.27 + 0.02

1.7 ± 0.4

6.3 ± 0.5

Y341A

0.40

0.06

3.2 ± 0.5

8.0 + 0.7

+

28.0

mmin-) 7.8

+ + + A

1.0

x

+ + + ±

Decamethonium

284C51

BW

1

1

1.5 4.0 6.0 36.0

8 100 100 833

10 113 80 1280

1.0 1.5 1.2

10 14 77

40 80 800

12.0

9

13

0.8 1.0

1.0 0.8

1 13

2

1.0

7

3

2

1.7

10

16

1.5

aWild type (WT) HuAChE IC50 values for edrophonium, propidium, decamethonium and BW284C51 ATCh at 0.5 mM was used in inhibition studies (for more details see Materials and methods).

are:

1.5, 1.5, 8.5 and 0.05 /4M, respectively.

3563

A.Shafferman et al.

striking effect on the reactivity towards edrophonium occurs when E202 is mutated. In this position even a conservative replacement (E202D) resulted in a 20-fold increase in the

IC50 value for edrophonium, as compared with wild type. Y337A mutant was 15-fold more resistant to inhibition by edrophonium while Y337F AChE was as sensitive as

001100

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0 0

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0

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0

0

0

v-0

0 0

[Decamethonium]i

0

0

0 0

[BW284C51] (j±Mi)

0

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%FLMI

Fig. 3. Inhibition of HuAChE and D74-HuAChE mutants by bisquaternary ligands. Enzyme preparations (5-20 ng/ml in 50 mM phosphate buffer pH 8) were preincubated for 20 min at 27°C with the indicated inhibitors; the reaction was initiated by adding ATCh to a final concentration of 0.5 mM. Wild type rHuAChE, open triangle; D74E, closed square; D74N, closed circle; D74G, open circle; D74K, closed triangle. AB

60

40

-I II GN

20/

0

E

_

D

E

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0

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F

E

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40/

20

0

.035

0.15

0.6

2.6

11

45

0.15

0.6

2.6

11

45

[ATChl (mM) Fig. 4. Dependence of the rate of hydrolysis of ATCh by HuAChE and its mutants on substrate concentration. Enzyme preparations (5-20 ng/ml in 50 mM phosphate buffer pH8) were reacted at 27°C with increasing concentrations (presented in loglo scale) of ATCh. In each of the panels the profile of the wild type HuAChE is depicted as a broken line. Panel A: D74 mutants. D74E, closed square; D74N, closed circle; D74G, open circle; D74K, closed triangle. Panel B: W286A mutant. Panel C: Y341A mutant. Panel D: Y337F, closed square; F338A, open circle. Panel E: Y337A mutant. Panel F: E202 mutants. E202Q, open circle; E202A, closed square; E202D, closed circle. 3564

Relay of substrate inhibition in acetylcholinesterase

wild type, suggesting that an aromatic amino acid at position 337 is required for better interaction of edrophonium with AChE. Mutations at W286, F338 or Y341 had a marginal effect on the reactivity towards edrophonium. Inhibition by propidium, a selective peripheral anionic site ligand (Taylor and Lappi, 1975), was most significantly affected by mutations at positions 74 and 286 (Figure 2 and

Table I). The D74E AChE mutant is almost as sensitive to inhibition by propidium as wild type AChE, and the rank order of susceptibility to inhibition by propidium is D74E > D74N > D74G > D74K. It thus appears that the charge at position 74 is a dominant factor affecting the interaction of propidium with AChE. The interaction of the two bisquaternary ligands,

Fig. 5. Molecular modeling of conformational changes associated with Y337. The active center region of HuAChE is viewed looking down into the 'gorge'; van der Waals surfaces for the C4 oxygen and C-3 hydrogen substituents of Y337 are indicated by dotted spheres. A. Aromatic residue of Y337 positioned in its native (XI = -155°; X2 = -11°) conformation in HuAChE. B. Aromatic residue of Y337 rotated (XI = 1430; X2 = 30°) into the gorge' space. In this location the aromatic residue interferes with access to Oy of serine-203. C. Aromatic residue of Y337 rotated as above in ACh-HuAChE complex, demonstrating the close contact between the C-3 hydrogen and the choline moiety.

3565

A.Shafferman et al.

decamethonium and BW284C51, with the various mutants is summarized in Table I. The only mutant exhibiting a wild type inhibition profile with these ligands was Y337F. The latter was also the only mutant to retain wild type susceptibility to edrophonium and propidium. With one exception (Y337A), any mutant that showed a reduced activity with either edrophonium or propidium also showed a decrease in affinity towards the bisquaternary ligands tested. Moreover, mutants defective in their interaction with both propidium and edrophonium (D74G, D74N and D74K) exhibited cooperative resistance to inhibition by the bisquatemary ligands (100- to 1000-fold higher than wild type; Figure 3 and Table 1). These observations are consistent with the fact that bisquatemary ligands, typified by decamethonium, are presumed to associate with both the active center and the peripheral site (Krupka, 1966; Quinn, 1987). Substitution in most of the positions targeted for mutagenesis produced enzymes with varying degrees of deviation in acetylthiocholine inhibition behavior (Figure 4). F338A and Y337F were the only mutants to exhibit the classical bell-shaped curves of hydrolysis rate versus concentration and practically overlap the native AChE profile (maximal activity at 0.6- 1.2 mM acetylthiocholine). Some mutants (W286A, Y341A and probably also D74E and E202Q) revealed a shift in the bell-shaped curve to higher concentrations of substrate, whereas other mutants (D74G, D74N, D74K, E202D, E202A and Y337A) showed no inhibition even at substrate concentrations as high as 90 mM (data not shown).

Discussion It has been postulated that the AChE active center contains a catalytic subsite as well as an anionic subsite responsible

for the binding of the positive charge of the choline moiety. It was identified by photoaffinity labeling studies that tryptophan 84 of TcAChE (W86 in HuAChE) is part of the anionic site in the active center (Kreienkamp et al., 1991) and according to the X-ray structure (Sussman et al., 1991) this residue is positioned in the active site gorge, 10 A from O-y of the catalytic serine (Figure 1). Others have also suggested that aromatic groups interact with quaternary ammonium ligands (Dougherty and Stauffer, 1990). Site directed labeling of the nicotinic ACh receptor suggests that aromatic amino acids form part of the ACh-binding region (Dennis et al., 1988; Abramson et al., 1989; Galzi et al., 1990). Thus, formation of 'anionic' binding site via aromatic residues may be an important feature of enzymes and receptors. The replacement of W86 in HuAChE with either an anionic residue, glutamate or an alanine residue yielded molecules that were not impaired in secretion and probably not in folding but failed to exhibit any detectable AChE activity. It is striking that out of 19 conserved HuAChE positions mutated to date, such a phenotype was manifested only by catalytic triad mutations: S203, H447 and E334 (Shafferman et al., 1992). We conclude therefore that W86 is indeed a critical element in the active center. Sussman et al. (1991) suggested on the basis of a model that involves docking of the ACh molecule, that the choline moiety makes close contact with the W84 of TcAChE. In our hands, docking of ACh onto a model of the HuAChE shows that the methyl groups of the quaternary ammonium are 3.64.0 A from the indole plane of W86 in the ACh -HuAChE

3566

complex. Recent crystallographic data for an edrophonium TcAChE complex show that the aliphatic substituents of the quatemary nitrogen of edrophonium are in a plane parallel to and within 4 A of the W84 indole ring of TcAChE (Sussman et al., 1992). Replacement of the aromatic residues at positions 337 and 338 with alanine did not abolish enzymatic activity and led to a mild reduction in catalytic efficiency (kcat, Table I). This is in accordance with their location at the middle/lower section of the 'gorge' (Figure 1) and with site directed affinity studies of TcAChE which suggested the involvement of these aromatic residues (330 and 331 in TcAChE) at the active center (Kieffer et al., 1986). Analysis of mutations involving E202, which is adjacent to the catalytic serine 203, supports some of the conclusions from an earlier site directed mutagenesis study (Gibney et al., 1990) performed on the analogous residue (E199) in TcAChE. Both studies demonstrate that these mutants exhibit higher Km values, suggesting a reduced affinity for the substrate, although the extent of the change is by far less pronounced in the HuAChE mutants. The ability to quantify the amount of enzyme involved in catalysis allowed us also to determine a 6- to 30-fold decrease in the apparent first order catalytic rate constant (kcat) of the E202 mutants, and a 17- to 140-fold decrease in the apparent bimolecular rate constant, as compared with the wild type enzyme. This could suggest that E202 mutants are much less effective in the acylation or deacylation catalytic steps. These observations can now be explained by the spatial orientation of E202 relative to the catalytic triad. The carboxyl group of E202 is within interaction distance (3.6 A) from N' of the imidazole ring of H447, as well as from the methylene adjacent to the trimethylammonium portion of ACh -HuAChE complex. This architecture may contribute to the stabilization of the protonated imidazolium facilitating proton transfer from serine 203 and thus formation of the acyl-enzyme intermediate. In addition to the active center esteratic and anionic subsites, it has been postulated that there is a peripheral anionic site on AChE (Changeux, 1966; Rosenberry, 1975; Quinn, 1987). A variety of uncompetitive AChE inhibitors, of which propidium is typical, are assumed to associate specifically with this binding site (Taylor and Lappi, 1975). Replacement of D74 as well as of W286, which is immediately adjacent to a peptide previously identified as part of a peripheral anionic site (Weise et al., 1990), produced AChE molecules with a lower affinity for propidium. We also found that mutations in these two positions generated AChE molecules in which inhibition by high substrate concentration is partially (W286A) or completely (D74N, D74G, D74K) eliminated. These results support kinetic studies that concluded that the peripheral and the substrate inhibition sites could overlap (Radic et al., 1991). It has been suggested that AChE is allosterically regulated by the binding of ACh to the peripheral site through conformational changes induced in the active center (Changeux, 1966). This would predict that substitution of one or more amino acids that participates in this signal transduction can alter the characteristics of both the active site and the peripheral site. Substitution at aspartate 74 clearly fulfils this prediction. Replacement of D74 (with asparagine, glycine or lysine) yields molecules with a lower affinity to both propidium and edrophonium. Furthermore these D74

Relay of substrate inhibition in acetylcholinesterase

mutated enzymes are also extremely resistant to bisquatemary ligands that presumably block both the peripheral and the active site. We propose that D74, which is located at the entrance of the 'gorge' (see Figure 1), could be an essential element in the relay of the conformational changes induced by ligand binding at the peripheral anionic site and in signalling these changes to the active site down at the bottom of the 'gorge'. According to our model the signal is transmitted from the peripheral anionic site through tryptophan 286 (and perhaps also through Y124 and Y72), located close to the rim of the 'gorge', to D74. Signal transduction from D74 down to the 'gorge' could involve Y341. Mutation in Y341, which can form a hydrogen bond with D74, produces an enzyme with a somewhat lower affinity for any of the tested ligands and one that is not effectively inhibited by high concentrations of acetylthiocholine. From Y341 the signal could eventually be transmitted to Y337. This is supported by the phenotype of the Y337A mutant which, in comparison with wild type, is more resistant to inhibition by edrophonium and is refractive to inhibition by high concentration of substrate. We further suggest, based on molecular modeling (Figure 5), that the allosteric changes initiated at the exterior can be translated into the inhibitory effect by a physical blockage of the active center through a conformational change in Y337. Such a change could prevent or interfere with the entry of substrate to an unoccupied active site and/or reduce the efficiency of the deacylation of the acyl -enzyme complex. Molecular mechanics calculation shows that the aryl moiety of Y337 can rotate as much as 620 (Figure SB) into the 'gorge' cavity without substantially altering the energy of the system (-- 2.0 Kcal/mol). A similar arrangement was in fact observed in the X-ray structure of a TcAChE -tacrine complex (Sussman et al., 1992). This rotation would place the aromatic side chain of Y337 in close contact with ACh docked at the active center and thus may block the formation of ACh -HuAChE complex (Figure SC). According to the proposed model, alanine at position 337 will not be able physically to block the entrance to the catalytic site, while an aromatic phenylalanine at this position (Y337F) could still exert the regulatory effect at high substrate concentrations, as is indeed the case. The observed impairment in substrate inhibition behavior in E202 HuAChE mutants and in the analogous mutants in TcAChE (Gibney et al., 1990) need not be related to the mechanism suggested above. It is possible that replacement of E202, which results in such significant changes in both Km and catalytic rates, actually disrupts the spatial arrangement in the active center to an extent that may interfere with a conformation change in Y337 or even override it. The crystal structure of the complex of TcAChE with edrophonium (Sussman et al., 1992) shows that the aromatic ring of F330 (analogous to Y337 of HuAChE) moves to make a better contact with the aromatic ring of the ligand. It is interesting to note that at high substrate concentrations, edrophonium binds to the active site with greater affinity (Radic et al., 1991), consistent with the idea of aromatic aromatic interaction, and also with the possibility that the aromatic side chain of Y337 responds conformationally to a signal initiated by substrate binding at the peripheral anionic site. The indole ring of residue W279 in TcAChE (analogous to W286 in HuAChE) displays a substantial movement upon binding of edrophonium or tacrine (Sussman et al., 1992).

Again, these experimental data clearly demonstrate the linkage between a conformational change in the active center and one in a distally located residue near the entrance of the 'gorge'. We note that the aromatic residues W286 and Y337 of HuAChE are replaced by alanine residues in human BChE (Prody et al., 1987; McTiernan, 1987), an enzyme devoid of substrate inhibition behavior. The physiological role of substrate inhibition may be an essential element in regulation of the residence time of ACh in the synapse. A sensor site, such as the peripheral anionic site on AChE surface, could trigger an inhibitory response to the initially high concentration of ACh near the presynaptic membrane. This would allow ACh to diffuse into the synaptic cleft and act on the receptor prior to its hydrolysis by AChE.

Materials and methods Construction of expression vectors for AChE and its mutants Plasmid construction, isolation of DNA fragments, cloning and bacterial transformation were performed essentially as described by Ausubel et al. (1987). Bipartite vectors derived from pEwCAT (Shafferman et al., 1992), expressing both human ache and the reporter cat genes were used to express the various AChE mutants in 293 human kidney cells. Mutagenesis was performed by DNA cassette replacement (Shafferman et al., 1987) into a series of HuAChE sequence variants (designated as Ewl, Ew4, Ew5 and Ew7) that conserve the wild type coding specificity, but carry new unique restriction sites (Shafferman et al., 1992). The cDNA spanning the amino acid targeted for mutagenesis was excised by cutting at the nearest restriction sites on the appropriate AChE cDNA variant and then replaced with a synthetic DNA duplex carrying the mutated codon. Mutagenesis was performed at position 74 [Asp codon GAC changed to GAG (Glu), GGG (Gly), AAC (Asn) or AAG (Lys)] and at position 86 [Trp codon TGG changed to GCC (Ala) or GAG (Glu)] by replacing the AccI-NruI DNA segment of pEw4; at position 202 [Glu codon GAG changed to GCC (Ala), CAG (Gln) or GAC (Asp)] by replacing the BstEII-SphI fragment of pEw4; at position 286 [Trp codon TGG to GCC (Ala)] by replacing the MtuI-PmlI fragment of pEw7; and at positions 337 [Tyr codon TAT changed to GCC (Ala) and TTC (Phe)], 338 [Phe codon TTT changed to GCC (Ala)] and 341 [Tyr codon TAC changed to GCC (Ala)] all by substitution of the BclI-NarI fragment in pEwS. All the synthetic DNA oligodeoxynucleotides ( - 60 nucleotides long) were prepared using an automatic Applied Biosystems DNA synthesizer. The sequences of all cloned synthetic DNAs were verified by the dideoxy sequencing method (USB Sequenase kit). Transient transfection and quantification of AChE CsCl purified plasmid preparations were used to transfect 293 cells using the calcium phosphate method (Wigler et al., 1977). At least two different clone isolates were tested for each plasmid construct. Transfection was carried on as described previously (Velan et al., 1991a,b) and cells were transferred 24 h after transfection to medium containing 10% AChE-depleted serum. Cells were incubated in 2 ml medium per 100 mm plate for 48 h. Medium was collected and assayed for AChE; medium from mock-transfected 293 cells served as a control. Cell lysates were assayed for intracellular AChE and CAT activity (Gorman et al., 1982). AChE activity was tested as described below and the mass of AChE protein was determined by a specific ELISA developed previously (Velan et al., 1991b; Shafferman et al., 1992). The levels of AChE protein produced by individual mutants (average of four transfections) were calculated by normalization to CAT activity.

Determination of AChE activity AChE activity in the medium of transfected cells was assayed according to Ellman et al. (1961). Standard assays were performed in the presence of 0.5 mM acetylthiocholine (ATCh), 50 mM sodium phosphate buffer, pH 8.0, 0.1 mg/ml BSA and 0.3 mM 5:5'-dithiobis(2-nitrobenzoic acid) (DTNB). The assay was carried out at 27°C and monitored using a Thermomax microplate reader (Molecular Devices). The effects of increased substrate concentrations on AChE activities were determined under similar assay conditions using increasing ATCh concentrations. Km values were obtained from Lineweaver-Burk plots and kcat calculations were based on ELISA quantifications. Interactions with inhibitors were monitored by determining residual activity after 20 min preincubation of rHuAChE or

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A.Shafferman et al. rHuAChE mutants with increasing concentrations of edrophorium, propidium, decamethonium or BW284C51 (all from Sigma).

Structural analysis and molecular graphics Analysis of the 3-D model of HuAChE was performed on a Silicon Graphics IRIS70/GT workstation, using the SYBYL modeling software (Trypos Inc.). The HuAChE model was constructed by comparative modeling methods (Barak et al., 1992), based on the known X-ray structure of TcAChE (Sussman et al., 1991). Structural refinement by molecular mechanics was done using the MAXMIN force field and zone refinement procedure Anneal, both included in SYBYL. Optimization of the ACh-HuAChE complex and structures with rotated aromatic side chain of Y337 included 23 amino acids.

Acknowledgements We would like to express our deep appreciation to Drs Sussman, Harel and Silman for sharing data prior to their publication. We thank Tamar Sery, Dana Stein, Gila Friedman and Nechama Zeliger for their excellent technical assistance and Dr S.Brimijoin for providing some of the monoclonal antibodies. This work was supported by the US Army Research and Development Command, Contract DAMD17-89-C-9117 to A.S.

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Substrate inhibition of acetylcholinesterase: residues affecting signal transduction from the surface to the catalytic center.

Amino acids located within and around the 'active site gorge' of human acetylcholinesterase (AChE) were substituted. Replacement of W86 yielded inacti...
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