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Molecular mode of interaction of plant amine oxidase with the mechanismâbased inhibitor 2âbutyneâ1,4âdiamine
- FrÃ©bort, Ivo, Å ebela, Marek, Svendsen, Ib, Hirota, Shun, Endo, Masaaki, Yamauchi, Osamu, Bellelli, Andrea, Lemr, Karel, PeÄ, Pavel
- European journal of biochemistry 2000 v.267 no.5 pp. 1423-1433
- absorption, active sites, amino acids, binding sites, crystal structure, enzyme inactivation, enzyme inhibition, high performance liquid chromatography, oxidation, peas, peptides, proteolysis, solvents
- 2âButyneâ1,4âdiamine (DABI) is a mechanismâbased inhibitor of copperâcontaining plant amine oxidases; the number of turnovers that leads to enzyme inactivation is ââ20. The product of DABI oxidation is a very reactive aminoallene that reacts with an essential nucleophilic group at the enzyme active site, forming a covalently bound pyrrole and producing an inactive enzyme. The inactivated enzyme shows a new absorption maximum at 295ânm and gives coloured derivatives with pâdimethylaminobenzaldehyde and pâdimethylaminocinnamaldehyde that are spectrally similar to the products of pyrrole treated with the above reagents. Resonance Raman spectra of the pâdimethylaminobenzaldehyde adduct of pyrrole and the inactivated enzyme show very high degree of similarity, supporting the idea that the product of inactivation is indeed a bound pyrrole. The bound pyrrole is formed already in the anaerobic step of the reaction, while the topa semiquinone radical is not affected, as shown by the EPR and stoppedâflow absorption measurements. Peptides containing the DABI binding site were obtained by proteolysis of inactivated enzyme, isolated by HPLC and analysed by amino acid sequencing and MS. The crystal structure of the amine oxidase from pea has been determined; inhibition is caused mainly by the highly reactive DABI product, 4âaminoâ2âbutynal, binding to a nucleophilic residue at the entrance to the substrate channel. As other DABI labelled peptides were also found and no free DABI product was detected by MS after complete inhibition of the enzyme, it is likely that the DABI product binds also to other solvent exposed nucleophilic residues on the enzyme surface.