Targeted oxidation of Torpedo californica acetylcholinesterase by singlet oxygen: identification of N-formylkynurenine tryptophan derivatives within the active-site gorge of its complex with the photosensitizer methylene blue

Abstract

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 (1)O(2) 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 Trp(84) and Trp(279), 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 (1)O(2) 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.

Figure 1

Singlet oxygen formation from methylene blue and its reaction with Trp. a) Formation of singlet oxygen generated by the irradiation of methylene blue and b) oxidation of a tryptophan residue to NFK and kynurenine by ¹O₂. (T₁: triplet state; ISC: intersystem crossing)

Figure 2

Cross-section through the active-site gorge of TcAChE in the MB/TcAChE complex. The MB molecule is seen stacked against Trp 279 in the peripheral anionic site (PAS). Also shown are Trp 84, in the catalytic anionic subsite of the active site (CAS), and the active-site serine, Ser 200. The entrance to the gorge is depicted at the top. The crystal structure of the MB/TcAChE complex has been deposited in the Protein Data Bank with access code 2W9I.

Figure 3

Immunological detection of NFK in TcAChE samples light-irradiated in the presence of MB. a) Coomassie blue staining and b) anti-NFK blotting of TcAChE-containing samples. The protein (1) was mixed with MB (2) in the dark or further irradiated for (3) 1, (4) 5, (5) 10 or (6) 20 minutes. Other lanes correspond to the reaction of TcAChE plus MB in the presence of inhibitors (7) in the dark or further irradiated for (8) 1, (9) 5, (10) 10 or (11) 20 minutes.

Figure 4

Deconvoluted MS/MS spectrum acquired from a parent ion of m/z 826.4³⁺, which corresponds in mass to tryptic peptide 270–289 + 32 Da. For the sake of clarity, not all identified fragment ions are labeled on the spectrum.

Figure 5

Deconvoluted MS/MS spectrum acquired from a parent ion of m/z 1063.4³⁺, which corresponds in mass to chymotryptic peptides 71–96 + 64 Da. For the sake of clarity, not all identified fragment ions are labeled on the spectrum.

Figure 6

Chromatograms of chymotryptic peptides which contain NFK derivatives from a) Trp 84 (m/z 1058.1³⁺) and b) Trp 279 (m/z 632.3²⁺), respectively. The signal intensity is indicated for the total ion current chromatogram. Analyses of samples containing TcAChE plus MB further irradiated for 20 minutes are indicated by dotted lines (without the inhibitors, ED and PR) and solid lines (with the inhibitors).