ANALYSIS OF THE ACTIVE-SITES OF COPPER TOPA QUINONE-CONTAINING AMINE OXIDASES FROM LATHYRUS-ODORATUS AND L-SATIVUS SEEDLINGS/

Amine oxidases from Lathyrus odoratus and L. sativus were isolated and , following a three step purification, gave pink coloured proteins (la mbda(max) 500 nm) which consisted of dimers of 72 kDa units each. Rabb it antiserum against the enzyme from L. odoratus cross-reacted with th e enzyme from L. sativus, N-Terminal amino acid sequences of both enzy mes show a high degree of similarity to other plant and microbial copp er-containing amine oxidases, Aliphatic diamines and some polyamines w ere the best substrates, whereas substrate analogues, copper complexin g agents and alkaloids were inhibitors of both amine oxidases, Differe ntial pulse polarography indicated the existence of a copper-quinone r edox system at the active site of the enzymes, Electron paramagnetic r esonance (EPR) spectra confirmed the presence of Cu(II) ions coordinat ed in a tetragonal ligand field, Mn(II) ions were clearly detected, wh ich are supposed partially to occupy another metal site in the enzyme. Spectrophotometric titrations with phenylhydrazines demonstrated one reactive carbonyl group per subunit of the enzymes. The pH shift of th e absorption maximum of p-nitrophenylhydrazones of the enzymes and res onance Raman spectroscopy strongly suggest topa quinone as the organic cofactor. (C) 1998 John Wiley & Sons, Ltd.