Trp82 and Tyr332 are involved in two quaternary ammonium binding domains of human butyrylcholinesterase as revealed by photoaffinity labeling with [3H]DDF.
Purified butyrylcholinesterase (BuChE) was photolabeled by [3H]-p-N, N-dimethylamino benzene diazonium ([3H]DDF) to identify the quaternary ammonium binding sites on this protein [Ehret-Sabatier, L. , Schalk, I., Goeldner, M., and Hirth, C. (1992) Eur. J. Biochem. 203, 475-481]. The covalent photoincorporation occurs with a stoichiometry of one mole of probe per mole of inactivated site and could be fully prevented by several cholinergic inhibitors such as tacrine or tetramethylammonium. After complete deglycosylation of the enzyme using N-glycosidase F, the alkylated protein was trypsinolyzed and the digests were analyzed by HPLC coupled to ES-MS. A direct comparison of tryptic fragments from labeled and unlabeled BuChE allowed us to identify the tryptic peptide Tyr61-Lys103 as carrying the probe. Purification of the labeled peptides by anion-exchange chromatography gave a major radioactive peak which was further fractionated by reversed-phase HPLC leading to three, well-resolved, radioactive peaks. Microsequencing revealed that two of these peaks contained an overlapping sequence starting at Tyr61, while the third peak contained a sequence extending from Thr315. Radioactive signals could be unambiguously attributed to positions corresponding to residues Trp82 and Tyr332. This labeling study establishes the existence of two different binding domains for quaternary ammonium in BuChE and exemplifies additional cation/pi interactions in cholinergic proteins. This work strongly supports the existence of a peripheral anionic site in BuChE, implying residue Tyr332 as a key element.
Nachon
F
F
Laboratoire de Chimie Bio-organique, UMR 7514 CNRS, Faculté de Pharmacie, Université Louis Pasteur Strasbourg, Illkirch, France.
Ehret-Sabatier
L
L
Loew
D
D
Colas
C
C
van Dorsselaer
A
A
Goeldner
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001881:Borates / Q000378:metabolism*; D002091:Butyrylcholinesterase / Q000737:chemistry / Q000378:metabolism*; D003979:Diazonium Compounds / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D019920:Photoaffinity Labels / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013552:Swine; D014316:Tritium; D014364:Tryptophan / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
D001881:Borates; D003979:Diazonium Compounds; D010446:Peptide Fragments; D019920:Photoaffinity Labels; C113457:p-(N-N-dimethylamino)-benzendiazonium fluoroborate; D014316:Tritium; D014443:Tyrosine; D014364:Tryptophan; D002091:Butyrylcholinesterase