Intermediates in the catalytic cycle of lentil (Lens esculenta) seedling copper-containing amine oxidase

Abstract

Spectrophotometry and rapid-scanning stopped-flow spectroscopy have been used to investigate the visible absorbance changes that occur in the course of the reduction of lentil (Lens esculenta) seedling amine oxidase by substrate. The catalytic cycle of the enzyme employs several intermediates but, owing to kinetic limitations, some of them were not identified in previous studies. In this study we have examined several substrates, either rapidly reacting (e.g. putrescine) or slowly reacting (e.g. gamma-aminobutanoic acid). Two forms of the enzyme, namely the Cu(I)-aminoresorcinol and quinone ketimine derivatives, whose characterization was elusive in previous studies, have been identified and assigned an optical spectrum. Moreover the reduced form of the enzyme is shown to be an equilibrium mixture of two species, the Cu(I)-semiquinolamine radical and Cu(II)-aminoresorcinol; these have been resolved by pH dependence and assigned spectra as well as a second-order rate constant for the reaction with oxygen. Thus the results presented here identify all the catalytic intermediates suggested by the chemical nature of the coenzyme and define their spectroscopic and reactivity properties.

Copper/topaquinone-containing amine oxidase from lentil seedlings and bovine plasma: Catalytic mechanism and energetic domains

“…In order to maximize the population of the quinone ketimine species in the catalytic cycle of LSAO, Floris and coworkers [17] used a poor substrate, -aminobutanoic acid, with k cat = 8. 3 10 -4 s -1 (k cat represents mol of substrate consumed/mol of active sites in 1 s).…”

“…The solution turns yellow as a result of the formation of new absorption bands centred at 464, 434 and 360 nm, indicative of the free radical intermediate species (Scheme 2, b) [24]. The extinction coefficients of reduced LSAO at pH 7 and 298 °K are: 464 = 7.1 mM -1 cm -1 ; 434 = 4.6 mM -1 cm -1 [17]. The electron transfer rate between Cu(II)-aminoresorcinol and the radical species is fast and involves a Cu(II) Cu(I) transition.…”

“…The electron transfer rate between Cu(II)-aminoresorcinol and the radical species is fast and involves a Cu(II) Cu(I) transition. The radical species so obtained is stable in anaerobiosis for many hours (t ½ = 36 h); after readmission of oxygen it releases H 2 O 2 and ammonia, thereby regenerating the Cu(II)-quinone species [4,17].…”

“…the "yellow" reduced enzyme is a mixture of Cu(I)-semiquinolamine and Cu(II)-aminoresorcinol [4,17]. The static spectrum of reduced LSAO is shown to be pH dependent [17], reflecting opposite effects of two titratable groups (with pKs = 5.7 and 7.9 respectively) on the equilibrium between the Cu(II)-aminoresorcinol and the Cu(I)-semiquinolamine.…”

“…The static spectrum of reduced LSAO is shown to be pH dependent [17], reflecting opposite effects of two titratable groups (with pKs = 5.7 and 7.9 respectively) on the equilibrium between the Cu(II)-aminoresorcinol and the Cu(I)-semiquinolamine. The latter has a maximum absorbance at 464, 434 and 360 nm at pH 7, which decreases both at acidic and alkaline pH values, and fades out at extreme pH values (5 and 10).…”