A Study on the Reactions of Plant Copper Amine Oxidase with C3 and C4 Aliphatic Diamines

The paper reports a study on the reactions of grass pea (Lathyrus sativus) amine oxidase (GPAO) with several aliphatic diamines. The influence of the chain length and of unsaturations in the molecules was examined. Kinetic measurements confirmed that trans-, i.e., (E)-2-butene-1,4-diamine (TDABE) and cis-, i.e., (Z)-2-butene-1,4-diamine (CDABE) could be classified as good substrates. Propane-1,3-diamine (DAP) and propene-1,3-diamine (DAPE) were only weakly oxidized, whereas 1,3-diamino-2-propanol (DAPL) was not utilized as a substrate. Contrary to the inactivator 2-butyne-1,4-diamine (DABI), DAPE was shown to be only a competitive inhibitor. DAP itself did not inhibit the catalytic activity. Irreversible inhibition of the activity occurred only after the incubation of GPAO with DABI; other diamines were without this effect. Differential pulse polarography and chromatofocusing confirmed that the aminoaldehyde product of DABI oxidation binds to the enzyme. Activity assay of pea aminoaldehyde dehydrogenase enabled us to detect the products of the oxidation of TDABE, CDABE, and DAP by GPAO. As the product of DAP oxidation, 3-aminopropanal (APAL) was detected by mass spectrometry and confirmed to be a potent noncompetitive inhibitor of GPAO. The absorption changes that occurred in the course of the reaction of GPAO with the diamines were investigated using rapid-scanning spectrophotometry. DABI, TDABE, CDABE, DAP, and DAPE reacted with GPAO providing characteristic maxima of the Cu(I)-semiquinolamine species that is formed in the catalytic cycle. The results presented here confirm that with the exception of DAPL, all the studied diamines could be classified as GPAO substrates, but only DABI can be considered as a mechanism-based inhibitor.