Characterization of Euphorbia characias latex amine oxidase

Abstract

A copper-containing amine oxidase from the latex of Euphorbia characias was purified to homogeneity and the copper-free enzyme obtained by a ligand-exchange procedure. The interactions of highly purified apo- and holoenzyme with several substrates, carbonyl reagents, and copper ligands were investigated by optical spectroscopy under both aerobic and anaerobic conditions. The extinction coefficients at 278 and 490 nm were determined as 3.78 x 10(5) M-1 cm-1 and 6000 M-1 cm-1, respectively. Active-site titration of highly purified enzyme with substrates and carbonyl reagents showed the presence of one cofactor at each enzyme subunit. In anaerobiosis the native enzyme oxidized one equivalent substrate and released one equivalent aldehyde per enzyme subunit. The apoenzyme gave exactly the same 1:1:1 stoichiometry in anaerobiosis and in aerobiosis. These findings demonstrate unequivocally that copper-free amine oxidase can oxidize substrates with a single half-catalytic cycle. The DNA-derived protein sequence shows a characteristic hexapeptide present in most 6-hydroxydopa quinone-containing amine oxidases. This hexapeptide contains the tyrosinyl residue that can be modified into the cofactor 6-hydroxydopa quinone.

Figure 1

SDS-PAGE pattern of ELAO. Lane a, ELAO; lane b (from top to bottom) ELAO with low standard proteins: phosphorylase B (97.4 kD), BSA (66 kD), ovalbumin (43 kD), carbonic anhydrase (31 kD), and trypsin inhibitor (20.1 kD).

Figure 2

Half-titration of ELAO with BHY. ELAO (24-nmol active sites) in 1 mL of 100 mm KPi buffer, pH 7.0, was treated with aliquots (5 μL = 1 nmol) of BHY. Spectra were recorded before (a) and after (m) (second spectra) each addition of BHY. Inset, Correlation between spectrum modification and inhibition of enzymic activity of ELAO by BHY. Absorbance readings were taken at 380 nm (•) and 5-μL portions of the reaction mixture were taken simultaneously for activity assay (▪).

Figure 3

A, Correlation between spectrum modification and inhibition of enzymatic activity of ELAO by PHY. For each aliquot (5 μL of 1-nmol PHY to 10-nmol ELAO active sites in 1 mL of 100 mm KPi buffer, pH 7.0) absorbance readings were taken at 430 nm (•) and 5-μL portions of the reaction mixture were taken from the cuvette for activity assay (▪). B, Correlation between spectrum modification and inhibition of enzymatic activity of ELAO by SCA. For each aliquot (5 μL of 2.5-nmol SCA to 17-nmol ELAO active sites in 1 mL of 100 mm KPi buffer, pH 7.0) absorbance readings were taken at 480 nm (*) and 345 nm (•), and 5 μL of the reaction mixture was removed from the cuvette for activity assay (▪).

Figure 4

Time course of the reaction of 18-nmol ELAO active sites with 2 mm DABA in 1 mL of 100 mm KPi buffer, pH 7.0, in anaerobic conditions, measured at 460 nm (•), 400 nm (▵), and 350 nm (▪).

Figure 5

UV-visible spectra of 50 μm kynuramine (a) and 50 μm 4-hydroxyquinoline (b) in 100 mm KPi buffer, pH 7.0.

Figure 6

Absorption spectra of copper-free ELAO upon reaction with kynuramine. The sample contained 11 μm copper-free enzyme under anaerobic conditions in 100 mm KPi buffer, pH 7.0. Spectrum a, Copper-free ELAO after addition of 160 μm kynuramine; spectrum b, spectrum a 5 min after the addition of CuCl₂ (200 μmol); spectrum c, spectrum a after 10 min. The dotted line represents the dilution effect after addition of CuCl₂. Inset, Spectrum of 11 μm ELAO copper-free enzyme.

Figure 7

Sequence of the ELAO (E) DNA compared with cDNA of LSAO (L) and PSAO (P). Circles indicate the Tyr that is modified to TPQ and two conserved His residues. Differences are underlined.