Confirmation of the presence of a Cu(II)/topa quinone active site in the amine oxidase from fenugreek seedlings

Abstract

Amine oxidase from etiolated seedlings of fenugreek (Trigonella foenum-graecum) has been isolated by a purification procedure involving three chromatographic steps. The homogeneous enzyme is of pink colour with a visible absorption maximum at 500 nm. The dimeric enzyme (2 x 75 kDa) is a slightly acidic protein (pl 6.8) containing 8% neutral Sugars, N-terminal amino acid sequence of the enzyme shows a high degree of similarity to other plant and microbial copper-containing amine oxidases. The best substrates of the enzyme are aliphatic diamines and some polyamines, whereas inhibitors are substrate analogues, copper complexing agents, some alkaloids and several other compounds. Spectrophotometric titrations with phenylhydrazines demonstrated one reactive carbonyl group per subunit of the enzyme and redox-cyclic quinone staining after native electrophoresis indicated the presence of a quinone cofactor. Differential pulse polarography showed the existence of a copper/quinone-containing active site. The resonance Raman spectroscopy and the pH-dependent shift of the absorption spectrum of the enzyme p-nitrophenylhydrazone confirm unambiguously the identity of the cofactor with topa quinone. EPR spectra of the enzyme are in accordance with those of tetragonal cupric complexes as known for other copper-containing amine oxidases. Besides the copper, Mn(II) ions were detected that partially occupy another metal site in the enzyme, but their catalytical importance is unlikely.