The principal role of AChE (acetylcholinesterase) is termination of impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter acetylcholine. The active site of AChE is near the bottom of a long and narrow gorge lined with aromatic residues. It contains a CAS (catalytic ‘anionic’ subsite) and a second PAS (peripheral ‘anionic’ site), the gorge mouth, both of which bind acetylcholine via π-cation interactions, primarily with two conserved tryptophan residues. It was shown previously that generation of 1O2 by illumination of MB (Methylene Blue) causes irreversible inactivation of TcAChE (Torpedo californica AChE), and suggested that photo-oxidation of tryptophan residues might be responsible. In the present study, structural modification of the TcAChE tryptophan residues induced by MB-sensitized oxidation was investigated using anti-N-formylkynurenine antibodies and MS. From these analyses, we determined that N-formylkynurenine derivatives were specifically produced from Trp84 and Trp279, present at the CAS and PAS respectively. Peptides containing these two oxidized tryptophan residues were not detected when the competitive inhibitors, edrophonium and propidium (which should displace MB from the gorge) were present during illumination, in agreement with their efficient protection against the MB-induced photo-inactivation. Thus the bound MB elicited selective action of 1O2 on the tryptophan residues facing on to the water-filled active-site gorge. The findings of the present study thus demonstrate the localized action and high specificity of MB-sensitized photo-oxidation of TcAChE, as well as the value of this enzyme as a model system for studying the mechanism of action and specificity of photosensitizing agents.
- acetylcholinesterase (AChE)
- active-site gorge
- mass spectrometry
- singlet oxygen
- site-specific photo-oxidation
The principle biological role of AChE (acetylcholinesterase) is the termination of impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter ACh (acetylcholine) . In keeping with its biological role, AChE possesses a very high specific activity, functioning at a rate approaching that of a diffusion-limited reaction . The toxicity of organophosphate nerve agents and insecticides is due to their potent inhibition of AChE . The first generation of drugs for the treatment of Alzheimer's disease are AChE inhibitors that act to overcome the cholinergic insufficiency resulting from degeneration of cholinergic nerve endings [4,5].
The electric organs of Torpedo californica and Electrophorus electricus are rich sources of AChE that is structurally homologous to that found in mammalian nerve and muscle . Unexpectedly for such a rapid enzyme, the crystal structure of TcAChE (T. californica AChE) revealed that its active site is near the bottom of a deep and narrow gorge lined by the rings of 14 conserved aromatic residues . It contains two subsites: a catalytic triad very similar to that found in other serine hydrolases, and a so-called CAS (catalytic ‘anionic’ site), which recognizes the quaternary group of ACh. A PAS (peripheral ‘anionic’ site) at the entrance to the active site gorge mediates substrate trapping . At both sites, recognition of ACh is primarily via π-cation interactions with conserved aromatic residues [9,10].
Recently, Weiner et al.  investigated the interaction of MB (Methylene Blue) with TcAChE. MB is a singlet oxygen (1O2)-generating photosensitizer that is also a strong reversible inhibitor of TcAChE in the dark [12,13]. The mechanism of generation of 1O2 by light irradiation of MB is shown in Figure 1(a). The principal amino acid residues in proteins oxidized by 1O2 are histidine, cysteine, methionine, tyrosine and tryptophan. Oxidation of tryptophan residues results in the formation principally of NFK (N-formylkynurenine) and kynurenine (Figure 1b). The MB–TcAChE complex was used as a model system for studying the chemical and structural consequences of photo-oxidation of residues within the active-site gorge [it is assumed, a priori, that the photochemical properties of MB in its complex with TcAChE, namely the life time of MB* (excited state) and the efficacy of energy transfer to oxygen are similar to its properties in its uncomplexed state].
Indeed, illumination of TcAChE in the presence of MB produces time- and concentration-dependent inactivation of the enzyme, attributable to 1O2. Addition of reversible inhibitors of TcAChE, such as ED (edrophonium), which interacts at the CAS , prior to sample illumination in the presence of MB, protects the enzyme from inactivation, suggesting that the 1O2 produced by MB is localized to the active-site gorge.
As mentioned above, TcAChE contains 14 highly conserved aromatic amino acid residues that line ~40% of its surface . Both the CAS and the PAS contain a tryptophan residue, Trp84 and Trp279 respectively (TcAChE numbering) . The crystal structure of the MB–TcAChE complex [14,15] (PDB code 2W9I) reveals a single MB molecule stacked against Trp279, at the top or the gorge, and orientated along the gorge axis towards the active site (Figure 2). Comparison of the fluorescence spectra of photo-inactivated TcAChE and of the native protein showed changes characteristic of tryptophan residue modification that are also correlated with loss of activity .
The present study was undertaken to characterize and identify the specific structural modifications produced in the tryptophan residues of TcAChE by MB photo-inactivation. We used a recently developed antiserum to NFK [16,17], an oxidation product of tryptophan and of tryptophan residues (Figure 2), in Western blot analysis of TcAChE samples that had been photo-oxidized in the presence of MB. A correlation was revealed between photo-inactivation of TcAChE and tryptophan oxidation to NFK. MS was then used to precisely identify which of the 14 tryptophan residues of TcAChE were oxidized to NFK in the photo-inactivated samples, as well as to identify other potential structural tryptophan modifications. It was revealed that MB-mediated photo-oxidation involves primarily Trp84, within the CAS, and Trp279, within the PAS.
Guanidine hydrochloride, DL-DTT (dithiothreitol; >99%), iodoacetamide (bioUltra, >99%) and PNGase F (from Elizabethkingia miricola, solution in 20 mM Tris/HCl, pH 7.5, 50 mM NaCl and 1 mM EDTA) were obtained from Sigma. Trypsin and chymotrypsin (from bovine pancreas, modified, sequencing grade) were obtained from Roche Molecular Biochemicals. The anti-NFK serum was collected from immunized New Zealand White rabbits (Harlan Bioproducts). All other chemicals were of analytical grade and were purchased from Sigma or Roche Molecular Biochemicals.
Purification and photo-oxidation of TcAChE samples were performed as described previously .
Protein gel electrophoresis and Western blot analysis
Aliquots (10 μg) of each sample were electrophoresed with SDS under reducing conditions (50 mM DTT) through duplicate 4–12% BisTris gels (Invitrogen). One gel was stained with Coomassie Blue, and the second gel was transferred on to a nitrocellulose membrane for Western blot analysis using a rabbit polyclonal antiserum against NFK at a 1:1000 dilution, essentially as described previously . Detection of NFK-containing proteins was performed using an Odyssey imager (LI-COR).
Lyophilized samples were solubilized in 100 mM phosphate buffer, pH 7.4, to a final concentration of 2 μM in TcAChE, and 5 μl aliquots were treated in a dark room for MS analysis. Samples were mixed with 5 M guanidine chloride for 30 min at 50°C and treated with 4.8 mM DTT for 30 min at 25°C, then 14.1 mM iodoacetamide was added and the sample was incubated for 30 min at 25°C. Samples were diluted to 0.1 pmol/μl with 100 mM Tris/HCl, pH 8.5, just prior to deglycosylation with PNGase overnight at 37°C, and finally digested with chymotrypsin or trypsin at a protein/enzyme ratio of 20:1 at pH 8.0 for 8 h at 37°C.
A Waters Q-TOF Premier mass spectrometer equipped with a nanoAcquity UPLC system and NanoLockspray source was used for the acquisition of the LC (liquid chromatography)–ESI (electrospray ionization)/MSe (elevated-energy mode) [18,19] and LC–ESI/MS/MS (tandem MS) data. Separations were performed using a 3 μm nanoAcquity Atlantis dC18 (100 μm×100 mm) column (Waters) at a flow rate of 300 nl/min. A 5 μm nanoAcquity Symmetry C18 (100 μm×20 mm) trapping column (Waters) was positioned in-line with the analytical column. Injections of 10 pmol of TcAChE digests were made on to the column. Peptides were eluted using a linear gradient from 98% solvent A [water/0.1% formic acid (v/v)] and 2% solvent B [acetonitrile/0.1% formic acid (v/v)] to 95% solvent B over 120 min. The mass spectrometer settings for MS analyses were a capillary voltage of 3.5 kV, a cone voltage of 30 V, a source temperature of 80°C and a collision energy of 4 eV. The mass spectra were recorded over a scan range of 100–2000 Da. MS data were acquired using a MSe scanning approach in which two sets of MS data were collected: (i) low-energy LC–MS data, comprising MS data for peptide precursors with a collision energy of 4 eV; and (ii) elevated energy MS data (15–35 eV ramp), which contains fragment ions. Ions of peptides identified in MSe were then targeted for MS/MS acquisition with a collision energy of 15–45 eV ramp. All MS/MS spectra were manually validated. For calibration, an external lock mass was used with a separate reference spray (LockSpray) using a solution of glu-fibrinopeptide B (300 fmol/μl) in water/acetonitrile 80:20 (v/v) with 0.1% formic acid and a mass of 785.8496 (2+). Semi-quantitative analyses of NFK derivatives were performed in triplicate, acquired in the MS-only mode, with injections of 10 μl of sample on to the column. LC–MS, LC–ESI/MSe and LC–ESI/MS/MS data analyses were performed using MassLynx version 4.0 (Waters) and ProteinLynx software supplied by the manufacturer.
Immunological detection of NFK in MB photo-oxidized TcAChE
TcAChE mixed with MB or MB plus the protein inhibitors ED and PR (propidium) was irradiated for 1, 5, 10 or 20 min as described previously . Additionally, samples consisting of TcAChE alone, TcAChE with MB, or TcAChE with MB, ED and PR were maintained in the dark. Samples were then subjected to SDS/PAGE electrophoresis for parallel examination by Coomassie Blue staining and anti-NFK Western blotting (Figure 3). The stained gel confirms that all lanes contained equal amounts of protein. This gel also shows that the lanes with the MB-containing protein subjected to the longest duration of illumination (Figure 3, lane 6), as well as all of the light-exposed samples containing MB and the two AChE inhibitors (Figure 3, lanes 8–11), contain distinct bands of approximately 38 and 28 kDa. However, the staining of these additional bands appears minor compared with the signal of TcAChE protein at 65 kDa, and the anti-NFK Western blotting indicates that these fragments do not contain any NFK residue. The anti-NFK Western blotting shows that, whereas the MB-containing protein subjected to the longest duration of illumination stained strongly for NFK (Figure 3, lane 6), the comparable sample containing the AChE inhibitors stained only very weakly at 65 kDa (Figure 3, lane 11).
MS identification of tryptophan residue oxidation products
Although Figure 3 shows that NFK accumulates in photo-oxidized TcAChE, Western blot data cannot define which specific tryptophan residue(s) has been modified to NFK. MS was therefore used to identify which of the 14 tryptophan residues in TcAChE were oxidized to kynurenine (tryptophan and 4 Da), hydroxy-tryptophan (tryptophan and 16 Da) and NFK (tryptophan and 32 Da) [20,21].
MSe scanning was used, in which each ion detected for a specific retention time was fragmented without the selection of a precursor ion [18,19]. This allowed the collection of two sets of MS data, the first consisting of low-energy LC–MS data, comprising MS data for peptide precursors, and the second as elevated- energy LC–MSe data containing fragment ions. These two sets of MSe data were then combined into ‘pseudo MS/MS’ spectra for each precursor ion. Because MSe scanning imparts a high sensitivity, almost complete sequence coverage was obtained for both chymotrypsin and trypsin digests of the protein. Specifically, 13 of the 14 tryptophan residues were detected, all but Trp58. More importantly, the LC–MSe results provided information concerning the two tryptophan residues, Trp84 and Trp279, localized in the active-site gorge and suspected to be the main targets of the singlet oxygen reaction .
Although 13 of the 14 tryptophan-containing peptides were detected by LC–MSe analysis, only four contained oxidative modifications of +16, +32, +48 and/or +64 Da after 60 min of irradiation in the presence of MB (Table 1). Comparable analysis of a control TcAChE sample did not detect the modifications found in peptides containing Trp84, Trp279 or Trp432/Trp435, indicating that the alterations in these peptides were due to irradiation in the presence of MB. However, in the case of Trp233, an equivalent peptide with the addition of 32 Da was detected in control samples and could be attributed to an artefact generated during sample preparation. As summarized in Table 1, the LC–MSe results showed the oxidation of tryptophan-containing peptides due to MB-mediated photo-oxidation of TcAChE. Nevertheless, despite the detection of these oxidized derivatives, fragmentation of the precursor ions in MSe was insufficiently informative to clearly identify tryptophan as the primary oxidized residue of the modified peptides. Indeed, the MSe data are a complex mixture of fragment ions that do not allow the assignment of daughter ions to a specific precursor. Moreover, it appears that several TcAChE methionine residues were easily oxidized during sample preparation for MS analysis because identically modified peptides were detected in control samples containing TcAChE. Because these data did not provide a clear distinction between oxidation on methionine residues and oxidation on tryptophan residues, LC–MS/MS experiments were performed targeting oxidized peptides detected in the MSe mode to disambiguate the localization of NFK derivatives in TcAChE.
Analysis of peptide containing Trp279
Figure 4 shows the MS/MS spectrum of the ion m/z 826.43+, which could be assigned to the protonated peptide 270–289 with an additional 32 Da. Upon CID (collision-induced dissociation), this precursor ion showed a fragmentation pattern similar to that of the corresponding unmodified peptide, and gave rise mainly to N-terminal b ions and C-terminal y ions [22,23]. The series b10–b13 corresponded to a shift in mass of an additional 32 Da, whereas the series b5–b9 remained identical when compared with the same series in the MS/MS spectrum of the unmodified peptide. These data allowed the assignment of a NFK residue localized on position 279 of TcAChE. This result was corroborated by MS/MS experiments targeting the precursor ion of the protonated peptide 268–289 with an additional 32 Da, detected at m/z 912.03+, which also showed the formation of an identical oxidized product localized on Trp279 (results not shown). Therefore, taken together, these results lead to the conclusion that Trp279 is oxidized into NFK in the presence of singlet oxygen.
Analysis of peptide containing Trp84
Ions corresponding in mass to protonated peptides 72–96, a segment that contains Trp84, were observed at m/z 1047.33+, 1052.63+, 1058.03+ and 1063.43+ (Table 1). These would correspond to the addition of 16, 32, 48 or 64 Da respectively, compared with the unmodified peptide. MS/MS spectra of precursor ions m/z 1047.33+ and 1052.63+ indicated oxidation of one or two methionine residues, Met83 and/or Met90, into sulfoxide (results not shown). As mentioned above, it appears that methionine residues were easily oxidized during MS sample preparation because similar artefacts were detected in control samples containing only TcAChE.
However, the CID of ions m/z 1058.03+ and 1063.43+ suggests a +32 Da oxidation localized on residue 84. As shown in Figure 5, the fragmentation pattern of the precursor ion m/z 1063.43+ showed the oxidation of Met90 into sulfoxide. Indeed, the series b20–b25 was detected with a shift in mass of 64 Da, whereas the daughter ion b18 was detected with an additional 48 Da when compared with the same series in the MS/MS spectrum of an unmodified peptide. MS/MS data suggested that this additional 48 Da was localized on Met83/Trp84 because the fragmentation pattern showed a non modified series b4–b9. Moreover, supplementary internal fragments which contain the Met83/Trp84 sequence, such as protonated 82–85, 77–86 and 74–84 fragments, were also detected with an additional 48 Da compared with the unmodified fragments. Therefore, even if the MS/MS spectrum of the precursor ion m/z 1063.43+ did not allow differentiation between Met83 and Trp84 modifications, these data strongly suggest the oxidation of both Met83 and Met90 into sulfoxide and the formation of an NFK derivative of tryptophan localized on position 84.
These results were further supported by MS/MS experiments targeting the precursor ion, detected at m/z 1058.13+, containing an additional 48 Da, which showed a similar fragmentation pattern (results not shown). More particularly, MS/MS data clearly indicated that Met90 was not oxidized, whereas daughter ions containing Met83/Trp84 showed a shift of 48 Da in mass when compared with the same series in the MS/MS spectrum of the unmodified peptide. Therefore these results lead to the conclusion that Trp84 is oxidized into NFK in the presence of singlet oxygen.
Analysis of peptide containing Trp432 and Trp435
Supplementary Figure S1 (at http://www.BiochemJ.org/bj/448/bj4480083add.htm) shows the MS/MS spectrum of the precursor ion m/z 870.13+, which could be assigned to the protonated peptide 422–442 with an additional 48 Da. Upon CID, this precursor ion gave rise to mainly N-terminal b ions and C-terminal y ions. Although the signal-to-noise ratio of this MS/MS spectrum was relatively low, the detailed fragmentation derived from series b8–b13 and y10–y11 clearly showed that only daughter ions which contained Trp432 were detected with a shift in mass of 32 Da, whereas Met436 could also be oxidized into sulfoxide with an additional 16 Da. Supplementary internal fragments which contained Trp432, such as the protonated 425–433 and 431–434 fragments, were also detected with an additional 32 Da compared with the unmodified fragments. It should be noted that fragmentation of the precursor ion m/z 870.13+ indicated that Trp435 was not oxidized. Therefore these results led to the conclusion that Trp432, but not Trp435, was oxidized to NFK by MB-mediated photo-oxidation.
Analysis of peptide containing Trp233
As mentioned above, trypsin digest of TcAChE detected the formation of a Trp233-containing peptide with an additional 32 Da, observed in MS spectra at m/z 1109.63+ (Table 1). However, this ion was also detected in control samples containing only the enzyme, and thus was not a result of MB photo-oxidation. Nevertheless, Trp233-containing peptides were also studied to identify artefacts induced by sample preparation for MS. The MS/MS spectrum of the ion m/z 1109.62+ can be assigned to the protonated peptide 222–242 with an additional 32 Da (Supplementary Figure S2 at http://www.BiochemJ.org/bj/448/bj4480083add.htm). Upon CID, the series y11–y17 corresponded to a shift in mass of an additional 32 Da, whereas the series y2–y9 remained identical when compared with the same series in the MS/MS spectrum of the unmodified peptide. These data allowed the assignment of an NFK residue of Trp233 of TcAChE, probably generated during sample preparation for MS.
Semi-quantification of NFK in samples with increasing photosensitization
It was previously reported that TcAChE irradiated in the presence of MB undergoes time- and concentration-dependent inactivation . Therefore additional experiments were performed to evaluate the relative amount of NFK residues compared with the unmodified tryptophan residues in relation to irradiation time of a TcAChE/MB mixture. Because chymotrypsin primarily hydrolyses the peptide bonds of tyrosine, phenylalanine and tryptophan residues, meaning that structural modifications of tryptophan residues interfere with the enzyme activity, trypsin digests were used for MS semi-quantification of tryptophan modifications. Experiments were performed in triplicate and acquired in the MS mode only. Briefly, chromatograms were extracted for each tryptophan residue-containing peptide ion previously identified, and LC peaks of corresponding m/z were integrated. It should be noted that all charge states of peptides detected in MS spectra were considered to obtain the total area of unmodified peptides (AU), peptides which contain methionine oxidized into sulfoxide (AS), and NFK-containing peptides (ANFK). The percentage of NFK derivatives (RNFK) was then calculated using the following equation: RNFK=ANF/(AU+AS+ANFK). The relative quantification of NFK derivatives was performed using TcAChE–MB samples irradiated for 20 and 30 min.
As previously mentioned, modified peptides containing Trp84 and Trp279 were not detected in the control samples containing only TcAChE, indicating that NFK residues were generated only upon MB-mediated photo-oxidation. More precisely, the oxidation rate of these two tryptophan residues increased with irradiation time. As shown in Table 2, 10% and 7.6% of Trp84 and Trp279 respectively were converted into NFK after 30 min of irradiation in the presence of MB. Despite the low rate of tryptophan residue oxidation, these results correlate with a loss of TcAChE activity, which decreases to only 16% of native TcAChE after 30 min of photo-oxidation.
It should be noted that MS analysis showed that the percentage of the NFK derivatives of the Trp233-containing peptide was constant and low (<0.5%). As previously mentioned, the oxidation of Trp233 was assumed to be an artefact generated during MS sample preparation because the oxidized derivative could be detected in the control sample containing only TcAChE. Consequently, Trp233-derivative artefacts generated for preparation of MS samples could be considered as negligible.
In contrast to the Trp84-, Trp279- and Trp233-containing peptides, quantification data for the Trp432 NFK derivative were unreliable. Because protonated Trp432-containing peptides were detected with only a low intensity in MS spectra, sizable errors would be introduced during integration of chromatographic peaks. Nevertheless, modified Trp432-containing peptides were not detected in control samples containing only the enzyme, and analysis of samples irradiated for 60 min in the presence of MB clearly shows conversion of Trp432 into NFK.
Effect of competitive inhibitors on the formation of NFK
Previous work showed that reversible competitive inhibitors of TcAChE protect the enzyme from photo-inactivation . Indeed, gorge-spanning ligands such as ED provided substantial protection against 1O2-mediated enzyme inactivation by binding within the active-site gorge of the protein. Therefore additional MS experiments were performed to measure the protective effect of the competitive inhibitors ED and PR on the oxidation of tryptophan residues to NFK.
In contrast with the TcAChE–MB sample, the addition of the reversible inhibitors strongly prevented the formation of NFK. Indeed, compared with the signal observed with the TcAChE–MB system, only a very small NFK signal was detected in the Trp279-containing peptide when inhibitors were present during MB photo-oxidation (Figure 6a). Comparable analysis of the Trp84-containing peptide found no NFK when ED and PR were added to the reaction mixture (Figure 6b). These results correlate well with protein activity measurements performed after 20 min of MB-mediated photo-oxidation. In the presence of ED and PR, TcAChE enzyme activity was reduced to 63% of control, whereas the parallel sample without these inhibitors had enzyme activity that was only 30% of control. This indicates that ED and PR prevent MB access to Trp84 and Trp279, efficiently protecting the enzyme from photo-inactivation caused by singlet oxygen.
A previous study  showed that MB, a strong reversible inhibitor of TcAChE in the dark, binds within the active-site gorge and that subsequent illumination of this mixture produces 1O2, which inactivates the enzyme via alteration of specific amino acid residues within the active-site gorge. This work also showed that TcAChE inhibitors, including ED, could protect TcAChE from MB-mediated photo-inactivation. Fluorescence spectroscopy revealed that photo-inactivation was accompanied by a decrease in the apparent tryptophan fluorescence signal and an increase in the apparent NFK fluorescence signal. Taken together, these data suggest that MB-mediated 1O2-generation in the TcAChE active-site gorge leads to the alteration of two tryptophan residues, Trp279 and Trp84, located at the entrance to the gorge and at the bottom of the gorge adjacent to the catalytic trio respectively.
The present study was undertaken to precisely identify the 1O2-altered tryptophan residues resulting from MB-mediated photo-oxidation and to characterize the specific tryptophan oxidation product. Initially, Western blot analysis (Figure 3) using highly specific anti-NFK antiserum [16,17] confirmed the spectroscopic results , detecting NFK in MB-photo-oxidized protein that was absent in native protein and substantially reduced in TcAChE irradiated in the presence of both MB and the inhibitors. Next, LC–MSe was used to identify and analyse 13 of the 14 tryptophan residue-containing peptides of TcAChE and to examine the oxidative modifications found in the photo-oxidized sample.
Possible modification of several residues, in addition to tryptophan and methionine, was considered in processing the MSe data, including histidine, tyrosine, cysteine and proline. However, no oxidation products of any of these residues were detected. Only four of the tryptophan residue-containing peptides showed oxidative modifications. Trp233 contained an identical modification (+32 Da) in both the photo-oxidized sample and the control native sample, and this alteration was therefore attributed to oxidation occurring during sample preparation. The other three peptides, those containing Trp84, Trp279 and Trp432/Trp435, were also subjected to LC–MS/MS experiments to determine the exact localization of oxidized derivatives on tryptophan residues. The deconvoluted MS/MS spectra derived from these experiments (Figures 4 and 5, and Supplementary Figures S1 and S2) clearly allow the assignment of NFK residues to Trp84, Trp279 and Trp432. Although the signal-to-noise ratio for the LC peak from the peptide containing Trp432 was too low for any semi-quantitative analysis to be performed, LC–MS analyses from the peptides containing Trp84 and Trp279 were sufficiently sensitive to allow the correlation between increasing irradiation in the presence of MB, increasing accumulation of NFK and decreasing enzyme activity. Furthermore, when TcAChE samples irradiated in the presence of MB were compared with those irradiated with MB in the presence of ED and PR, the latter showed little or no tryptophan oxidation of Trp279 and Trp84.
The distance travelled by 1O2, R, will depend on the diffusion coefficient, D, and on its lifetime, τ : R2=6 Dτ. The values found experimentally for diffusion of 1O2 in cells, D ~2–4×10−6 cm2/s and τ~10 μs , predict R~500 Å (1 Å=0.1 nm), much larger than the radius of gyration of the catalytic subunit of TcAChE (~50 Å) or the length of its active-site gorge (15–20 Å) . However, once generated in a cellular or protein environment, 1O2 is unlikely to travel far due to the presence of natural quenchers; thus, its primary reactions should occur close to its site of generation. The distances between MB and the 14 tryptophan residues in TcAChE are listed in Table 3. The MS data revealed apparent oxidation by 1O2 of only four tryptophan residues, all conserved. Of these, Trp233 was modified to an equal extent in the dark and under illumination, and Trp432 displayed only a very low level of modification. Only Trp84 and Trp279, both within the active-site gorge, were extensively modified to their NFK degradation products, and the data displayed in Figure 6 clearly show that in the presence of competitive inhibitors that displace MB from the gorge, their modification is completely prevented. Trp279 is obviously the closest residue, stacked against MB. Trp84, which is in the ‘anionic’ subsite of the active site and interacts with the quaternary group of ACh, and Trp233, which is on the surface of the acyl pocket, are at about the same distance, 13.8 and 13.1 Å respectively. Although the indole ring of Trp84 is fully exposed to the active-site gorge, in the case of Trp233, examination of the crystal structure of TcAChE reveals that, whereas the phenyl moiety of the indole ring points towards the gorge, the 5-membered ring, the putative target of 1O2, is not exposed . Of the remaining residues, Trp432 is the closest, and indeed the peptide containing it is modified, albeit marginally. Restriction of significant oxidative damage to Trp84 and Trp279 in TcAChE is in keeping with the proposal of a facilitated diffusion pathway for 1O2 along a water-filled channel in the fluorescent protein KillerRed . Indeed, the aqueous channel seen in KillerRed is reminiscent of the active-site gorge of TcAChE.
These results have confirmed the formation of NFK in photo-oxidized TcAChE and identified the two major sites of oxidation to be on Trp84 and Trp279, both involved in the substrate binding in the active-site gorge of the enzyme. Despite evidence from previous work  that MB-mediated photo-inactivation produces only a modest perturbation of the TcAChE native structure, oxidation of these two tryptophan residues within the active-site gorge has a profound effect on enzyme activity. The specificity of the reaction can be explained by the localized action of the singlet oxygen inside the gorge mouth of the enzyme due to the specific binding of MB. Therefore the present study confirms that TcAChE is a valuable model for understanding the molecular basis of local photo-oxidative damage with selective modifications of the protein . The data obtained are pertinent to our understanding of the mode of action of photosensitizers in PDT (photodynamic therapy)  and the model using the TcAChE–MB system would be particularly valuable in studies devoted to related areas. Although the technique is clinically applied to treat various diseases such as macular degeneration, pathologicalmyopia, oesophagus, lung and skin cancer , understanding of the cytotoxic action of PDT at the molecular level is important both for development of more selective photosensitizers and for enhancement of their therapeutic efficacy.
Mathilde Triquigneaux performed MS experiments and wrote the paper. Marilyn Ehrenshaft performed protein gel electrophoresis and Western blotting experiments, and wrote the paper. Esther Roth performed the purification and photo-oxidation of TcAChE. Yakov Ashani performed experiments by X-ray and provided distances of the MB-TcAChE complex. Israel Silman, Lev Weiner, Ronald Mason and Leesa Deterding designed the experiments, contributed to writing the paper and provided financial support for the research.
This work was supported by the Intramural Research Program of the NIH (National Institutes of Health), National Institutes of Environmental Health Sciences [projects ES050139 and ES050171].
We thank Mary J. Mason and Dr Ann Motten for their editing of the paper prior to submission. We thank Joel Sussman, Harry Greenblatt and Moshe Ben-David for valuable discussions and help with the graphics.
Abbreviations: ACh, acetylcholine; AChE, acetylcholinesterase; CAS, catalytic ‘anionic’ site; CID, collision-induced dissociation; DTT, dithiothreitol; ED, edrophonium; ESI, electrospray ionization; LC, liquid chromatography; MB, Methylene Blue; MSe, combined low and elevated-energy mode MS; MS/MS, tandem MS; NFK, N-formylkynurenine; PAS, peripheral ‘anionic’ site; PDT, photodynamic therapy; PR, propidium; TcAChE, Torpedo californica AChE
- © The Authors Journal compilation © 2012 Biochemical Society