Tài liệu Báo cáo khoa học: 1,5-Diamino-2-pentyne is both a substrate and inactivator of plant copper

1,5-Diamino-2-pentyne is both a substrate and inactivator of plant

copper amine oxidases

Zbyneˇ k Lamplot1

, Marek Sˇ ebela1

, Michal Malonˇ

2

, Rene´ Lenobel2

, Karel Lemr3

, Jan Havlisˇ

4

, Pavel Pecˇ

1

,

Chunhua Qiao5 and Lawrence M. Sayre5

1

Department of Biochemistry, 2

Laboratory of Growth Regulators, and 3

Department of Analytical Chemistry, Faculty of Science,

Palacky´ University, Olomouc, Czech Republic; 4

Department of Analytical Chemistry, Faculty of Science, Masaryk University, Brno,

Czech Republic; 5

Department of Chemistry, Case Western Reserve University, Cleveland, OH, USA

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 pre￾vented 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. Pro￾longed incubations of GPAO or OVAO with DAPY resul￾ted in the appearance of a yellow–brown chromophore

(kmax ¼ 310–325 nm depending on the working buffer).

Excitation at 310 nm was associated with emitted fluores￾cence with a maximum at 445 nm, suggestive of extended

conjugation. After dialysis, the color intensity was substan￾tially decreased, indicating the formation of a low molecular

mass secondary product of turnover. The compound pro￾vided positive reactions with ninhydrin, 2-aminobenzalde￾hyde and Kovacs’ reagents, suggesting the presence of an

amino group and a nitrogen-containing heterocyclic struc￾ture. The secondary product was separated chromato￾graphically 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 1

H- and 13C-NMR

experiments. This structure suggests a lysine modification

chemistry that could be responsible for the observed inacti￾vation.

Keywords: amine oxidase; diamine; mechanism-based inhi￾bition; nuclear magnetic resonance; oxidation.

Copper-containing amine oxidases (CAOs, EC 1.4.3.6) play

a crucial role in the metabolism of primary amines. These

enzymes are widely distributed in nature [1]. In micro￾organisms, CAOs have a nutritional role in the utilization of

primary amines as the sole nitrogen and carbon source. In

mammals and plants, CAOs appear to be tissue specific, and

are implicated in wound healing, detoxification, cell growth,

signaling and apoptosis [1]. The oxidative deamination of

amine substrates catalyzed by CAOs yields the correspond￾ing aldehydes with the concomitant production of hydrogen

peroxide and ammonia [2].

The reaction proceeds through a transamination mech￾anism mediated by an active site cofactor topaquinone. The

cofactor is derived from the post-translational self-process￾ing of a specific tyrosine residue that requires both active site

copper and molecular oxygen [2]. The key step in the

oxidative deamination is conversion of the initial substrate

Schiff base (quinoimine) to a product Schiff base (quino￾aldimine) facilitated by Ca proton abstraction via a

conserved aspartate residue acting as a general base at the

active site [3]. This step is followed by hydrolytic release of

the aldehyde product and the reduced cofactor is finally

reoxidized by molecular oxygen with the release of H2O2

and NH4

+. The reduced topaquinone exists in two forms.

The first is an aminoresorcinol derivative coexisting with

Cu(II), which is in equilibrium with the second form, Cu(I)-

semiquinolamine radical [3]. The role of copper in the

reoxidation step has not been sufficiently elucidated for

Correspondence to M. Sˇebela, Department of Biochemistry, Faculty of

Science, Palacky´ University, Sˇlechtitelu˚ 11, CZ-783 71 Olomouc,

Czech Republic. Fax: + 420 5856 34933; Tel.: + 420 5856 34927;

E-mail: [email protected]

Abbreviations: ABA, 2-aminobenzaldehyde; ACA, 6-aminocaproic

acid; BEA, 2-bromoethylamine; CAO, copper-containing amine

oxidase; DABY, 1,4-diamino-2-butyne; DAPY, 1,5-diamino-2-pen￾tyne; DDD, 3,5-diacetyl-2,6-dimethyl-1,4-dihydropyridine; DMAB,

4-(dimethylamino)benzaldehyde; DMAC, 4-(dimethylamino)cinna￾maldehyde; ESI, electrospray ionization; GPAO, grass pea (Lathyrus

sativus) amine oxidase; HABA, 2-(4-hydroxyphenylazo)benzoic acid;

IT, ion trap; LSAO, lentil (Lens esculenta) amine oxidase; MALDI,

matrix-assisted laser desorption/ionization; OVAO, sainfoin

(Onobrychis viciifolia) amine oxidase; PSAO, pea (Pisum sativum)

seedling amine oxidase; PSD, post source decay; Q, quadrupole;

TNBS, 2,4,6-trinitrobenzenesulfonic acid.

Enzyme: copper-containing amine oxidase (EC 1.4.3.6).

Note: A website is available at http://prfholnt.upol.cz/biochhp

(Received 24 August 2004, revised 23 September 2004,

accepted 13 October 2004)

Eur. J. Biochem. 271, 4696–4708 (2004)
FEBS 2004 doi:10.1111/j.1432-1033.2004.04434.x