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.
Skip Nav Destination
Article navigation
November 2012
-
Cover Image
Cover Image
- PDF Icon PDF LinkFront Matter
- PDF Icon PDF LinkTable of Contents
- PDF Icon PDF LinkEditorial Board
Research Article|
October 18 2012
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
Mathilde M. Triquigneaux;
Mathilde M. Triquigneaux
1
*Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, DHHS, PO Box 12233 MD F0-03, Research Triangle Park, NC 27709, U.S.A.
1To whom correspondence should be addressed (email triquigneauxm@mail.nih.gov).
Search for other works by this author on:
Marilyn Ehrenshaft;
Marilyn Ehrenshaft
*Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, DHHS, PO Box 12233 MD F0-03, Research Triangle Park, NC 27709, U.S.A.
Search for other works by this author on:
Esther Roth;
Esther Roth
†Department of Neurobiology, Weizmann Institute of Science, Rehovoth, 76100, Israel
Search for other works by this author on:
Israel Silman;
Israel Silman
†Department of Neurobiology, Weizmann Institute of Science, Rehovoth, 76100, Israel
Search for other works by this author on:
Yakov Ashani;
Yakov Ashani
†Department of Neurobiology, Weizmann Institute of Science, Rehovoth, 76100, Israel
Search for other works by this author on:
Ronald P. Mason;
Ronald P. Mason
*Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, DHHS, PO Box 12233 MD F0-03, Research Triangle Park, NC 27709, U.S.A.
Search for other works by this author on:
Lev Weiner;
Lev Weiner
‡Department of Chemical Research Support, Weizmann Institute of Science, Rehovoth, 76100, Israel
Search for other works by this author on:
Leesa J. Deterding
Leesa J. Deterding
§Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, DHHS, PO Box 12233 MD F0-03, Research Triangle Park, NC 27709, U.S.A.
Search for other works by this author on:
Publisher: Portland Press Ltd
Received:
June 25 2012
Revision Received:
August 09 2012
Accepted:
August 13 2012
Accepted Manuscript online:
August 13 2012
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2012 Biochemical Society
2012
Biochem J (2012) 448 (1): 83–91.
Article history
Received:
June 25 2012
Revision Received:
August 09 2012
Accepted:
August 13 2012
Accepted Manuscript online:
August 13 2012
Citation
Mathilde M. Triquigneaux, Marilyn Ehrenshaft, Esther Roth, Israel Silman, Yakov Ashani, Ronald P. Mason, Lev Weiner, Leesa J. Deterding; 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. Biochem J 15 November 2012; 448 (1): 83–91. doi: https://doi.org/10.1042/BJ20120992
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Captcha Validation Error. Please try again.