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Analysis of the active sites of copper/topa quinone‐containing amine oxidases from Lathyrus odoratus and L. sativus seedlings
Author(s) -
Šebela Marek,
Luhová Lenka,
Frébort Ivo,
Faulhammer Heinz G.,
Hirota Shun,
Zajoncová Ludmila,
Stužka Václav,
Peč Pavel
Publication year - 1998
Publication title -
phytochemical analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.574
H-Index - 72
eISSN - 1099-1565
pISSN - 0958-0344
DOI - 10.1002/(sici)1099-1565(199809/10)9:5<211::aid-pca407>3.0.co;2-x
Subject(s) - chemistry , quinone , amine gas treating , enzyme , amine oxidase , copper , cofactor , electron paramagnetic resonance , redox , stereochemistry , biochemistry , organic chemistry , physics , nuclear magnetic resonance
Abstract Amine oxidases from Lathyrus odoratus and L. sativus were isolated and, following a three step purification, gave pink coloured proteins (λ max 500 nm) which consisted of dimers of 72 kDa units each. Rabbit antiserum against the enzyme from L. odoratus cross‐reacted with the enzyme from L. sativus. N‐Terminal amino acid sequences of both enzymes show a high degree of similarity to other plant and microbial copper‐containing amine oxidases. Aliphatic diamines and some polyamines were the best substrates, whereas substrate analogues, copper complexing agents and alkaloids were inhibitors of both amine oxidases. Differential pulse polarography indicated the existence of a copper–quinone redox system at the active site of the enzymes. Electron paramagnetic resonance (EPR) spectra confirmed the presence of Cu(II) ions coordinated in a tetragonal ligand field. Mn(II) ions were clearly detected, which are supposed partially to occupy another metal site in the enzyme. Spectrophotometric titrations with phenylhydrazines demonstrated one reactive carbonyl group per subunit of the enzymes. The pH shift of the absorption maximum of p ‐nitrophenylhydrazones of the enzymes and resonance Raman spectroscopy strongly suggest topa quinone as the organic cofactor. © 1998 John Wiley & Sons, Ltd.