title: 1,5-diamino-2-pentyne is both a substrate and inactivator of plant copper amine oxidases.
abstract: 1,5-diamino-2-pentyne (DAPY) was found to be a weak substrate of grass pea (Lathyrus sativus, GPAO) and sainfoin (Onobrychis viciifolia, OVAO) amine oxidases. Prolonged incubations, however, resulted in irreversible inhibition of both enzymes. For GPAO and OVAO, rates of inactivation of 0.1-0.3 min(-1) were determined, the apparent KI values (half-maximal inactivation) were of the order of 10(-5) m. DAPY was found to be a mechanism-based inhibitor of the enzymes because the substrate cadaverine significantly prevented irreversible inhibition. The N1-methyl and N5-methyl analogs of DAPY were tested with GPAO and were weaker inactivators (especially the N5-methyl) than DAPY. Prolonged incubations of GPAO or OVAO with DAPY resulted in the appearance of a yellow-brown chromophore (lambda(max) = 310-325 nm depending on the working buffer). Excitation at 310 nm was associated with emitted fluorescence with a maximum at 445 nm, suggestive of extended conjugation. After dialysis, the color intensity was substantially decreased, indicating the formation of a low molecular mass secondary product of turnover. The compound provided positive reactions with ninhydrin, 2-aminobenzaldehyde and Kovacs' reagents, suggesting the presence of an amino group and a nitrogen-containing heterocyclic structure. The secondary product was separated chromatographically and was found not to irreversibly inhibit GPAO. MS indicated an exact molecular mass (177.14 Da) and molecular formula (C10H15N3). Electrospray ionization- and MALDI-MS/MS analyses yielded fragment mass patterns consistent with the structure of a dihydropyridine derivative of DAPY. Finally, N-(2,3-dihydropyridinyl)-1,5-diamino-2-pentyne was identified by means of 1H- and 13C-NMR experiments. This structure suggests a lysine modification chemistry that could be responsible for the observed inactivation.
authors.affiliation: Department of Biochemistry, Faculty of Science, Palacky University, Olomouc, Czech Republic.
authors.forename: Zbynek
authors.initials: Z
authors.lastname: Lamplot
authors.forename: Marek
authors.initials: M
authors.lastname: Sebela
authors.forename: Michal
authors.initials: M
authors.lastname: Malon
authors.forename: René
authors.initials: R
authors.lastname: Lenobel
authors.forename: Karel
authors.initials: K
authors.lastname: Lemr
authors.forename: Jan
authors.initials: J
authors.lastname: Havlis
authors.forename: Pavel
authors.initials: P
authors.lastname: Pec
authors.forename: Chunhua
authors.initials: C
authors.lastname: Qiao
authors.forename: Lawrence M
authors.initials: LM
authors.lastname: Sayre
mesh_terms: D000480:Alkynes / Q000378:metabolism / Q000494:pharmacology*; D006631:Amine Oxidase (Copper-Containing) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003124:Colorimetry; D003959:Diamines / Q000378:metabolism / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D027807:Lathyrus / Q000201:enzymology*; D010084:Oxidation-Reduction; D013057:Spectrum Analysis / Q000379:methods; D013379:Substrate Specificity
chemical_list: C501748:1,5-diamino-2-pentyne; D000480:Alkynes; D003959:Diamines; D004791:Enzyme Inhibitors; D006631:Amine Oxidase (Copper-Containing)