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 K I values (half-maximal inactivation) were of the order of 10DAPY was found to be a mechanism-based inhibitor of the enzymes because the substrate cadaverine significantly prevented irreversible inhibition. The N 1 -methyl and N 5-methyl analogs of DAPY were tested with GPAO and were weaker inactivators (especially the N 5 -methyl) than DAPY. Prolonged incubations of GPAO or OVAO with DAPY resulted in the appearance of a yellow-brown chromophore (k max ¼ 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 (C 10 H 15 N 3 ). 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 1 H-and 13 C-NMR experiments. This structure suggests a lysine modification chemistry that could be responsible for the observed inactivation.

An important lysine residue in copper/quinone‐containing amine oxidases

“…A nucleophilic residue has been shown with certainty to be involved in the inhibition mechanism of AOs during the oxidation of 1,4‐diamino‐2‐butyne (DABY) [13,14], 1,5‐diamino‐2‐pentyne [15], the aromatic monoamine tyramine [16], and other selective AO inhibitors [17]. Although the involvement of a lysine has been postulated [14,17], compelling evidence has not been presented.…”

“…An important method in studying the structure-function of an enzyme is to find specific inhibitors and follow their effects. Our interest in the present study is in the mechanism-based inhibitor DABY, for the following two reasons: (a) the inhibitor has been found to be a suicide substrate for plant copper AO (Cu-AO) from pea seedlings [14] and grass pea [15], and for mammalian AOs from pig kidney [40] and from beef serum [17]; and (b) it has been postulated that the irreversible inhibition of all enzymes involves an intermediate aminoallenic compound that forms covalently bound pyrrole in the reaction with a nucleophile at the active site.…”

Mechanism-Based Inhibitors of Cytokinin Oxidase/Dehydrogenase Attack FAD Cofactor

“…They undergo turnover-dependent conversion to electrophilic products capable of covalent binding to an active-site nucleophile, resulting in enzyme inactivation. 14 Mechanism-based inhibitors are characterized by both substrate saturation kinetics and pseudo-firstorder time-dependent irreversible inactivation. The enzyme can be protected from inactivation by the addition of natural substrates or competitive inhibitors.…”