The reaction mechanism of copper amine oxidase: detection of intermediates by the use of substrates and inhibitors

Abstract

Intermediate states in the catalytic mechanism of lentil copper amine oxidase have been investigated by ESR and optical spectroscopy. Using highly purified apo- and holoenzyme in combination with a poor substrate and a range of inhibitors, under both aerobic and anaerobic conditions, the single steps of the reaction mechanism can be slowed down or 'frozen' completely. In this way, a sequence of six intermediate species in the catalytic cycle has been established. Oxidative deamination of p-(dimethylamino)benzylamine is 5 x 10(5) times slower than for putrescine; the rate-limiting step is shown to be release of the aldehyde product. This process is not affected in the apoenzyme, but subsequent intramolecular electron transfer to form the characteristic free radical intermediate is completely blocked, and the apoenzyme is trapped as an aminoresorcinol species. Classic hydrazine and hydrazide inhibitors bind to the 6-hydroxydopa cofactor in the same way as active substrates, but rearrangements lead to formation of stable intermediate adducts at the step preceding release of aldehyde. The semicarbazide-6-hydroxydopa adduct is shown to bind simultaneously to Cu(II), providing the first direct evidence for localization of 6-hydroxydopa close to the copper site.

Characterization of Euphorbia characias Latex Amine Oxidase1

“…Together with the formation of the quinolaldimine, a new band centered at 400 nm appeared. This band was assigned to the protonated tautomeric form of the quinolaldimine, as reported for DABA oxidation by LSAO (Medda et al, 1995b). Under anaerobic conditions this species decayed slowly, in parallel with formation of the yellow radical intermediate (Fig.…”

“…The catalytic mechanism of amine oxidases has been reported previously (Medda et al, 1995b;Steinebach et al, 1996). The amine substrate binds to the organic cofactor of the resting oxidized enzyme Cu(II)-TPQ to form a Schiff base, Cu(II)-quinone ketimine; both of these forms are assumed to give the broad 498-nm absorption band.…”

“…Amine oxidases are homodimers of 70-to 95-kD subunits. Each subunit contains a tightly bound Cu(II) center that is essential for the enzyme redox activity (Dooley et al, 1991;Medda et al, 1995b), and TPQ is formed by a posttranslational modification from a tyrosinyl residue in a copperdependent, autocatalytic reaction (Janes et al, 1990;Brown et al, 1991;Cai and Klinman, 1994;Matsuzaki et al, 1994;Choi et al, 1995;Nakamura et al, 1996;Mure and Tanizawa, 1997).…”

“…The aromatic aldehyde production from benzylamine was measured by the A 250 increase using an ⑀ 250 of 12,800 m Ϫ1 cm Ϫ1 (Neumann et al, 1975). The aldehyde production from DABA was measured by the A 350 increase using an ⑀ 350 of 31,200 m Ϫ1 cm Ϫ1 (Ehrlich's reagent; Medda et al, 1995b). The reaction with kynuramine was followed photometrically at 326 and 314 nm (formation of 4-hydroxyquinoline) or at 356 and 255 nm (disappearance of kynuramine; Padiglia et al, 1997).…”

“…When putrescine was added to ELAO in anaerobiosis, the broad absorption band at 490 nm disappeared instantaneously, indicating the rapid conversion of the TPQ cofactor to a bleached species, presumably the quinolaldimine. Then the solution turned yellow as a result of the formation of new absorption bands centered at 430 and 460 nm, indicative of a free radical intermediate species generated upon reaction in the absence of oxygen (Finazzi Agró et al, 1984;Dooley et al, 1987;Medda et al, 1995b). Precisely 20 nmol of Figure 2.…”