Active site of copper oxidases

Abstract

Copper amine oxidases catalyse the oxidative deamination of biological amines according to the following equation: RCH 2NH 2 + O 2 + H 2O → RCHO + H 2O 2 + NH 3 It should be recalled that another class of amine oxidases was described. They contain FAD as a prosthetic group, but will not be discussed here. All copper amine oxidases have molecular weight higher than 100,000: some of them appear to contain two equivalent subunits which are probably not linked by disulfide bridges [1,2]. All these enzymes contain at least two copper atoms and one carbonyl group per molecule. The nature of the carbonyl cofactor is a controversial issue: many data suggest that it could be identified as pyridoxal-5-phosphate [1]. Electron spin resonance (ESR) spectra show that the two copper atoms are in an equivalent environment and in the cupric state [3, 4]. The spectrum is typical of Cu(II) in a complex of tetragonal symmetry [3]. the use of 15N labeled putrescine demonstrated that the substrate is not directly bound to copper [3]. Magnetic resonance studies [3, 5] and the comparison of the absorption and ESR spectra of the bovine enzyme with a model complex [6] suggest that in these enzymes at least two nitrogen atoms and 2 oxygen (from water) are coordinated around the copper ion. When copper amine oxidases were treated with phenyllhydrazine, 1 carbonyl group/mol was titrated [1] and an enzymatically inactive irreversible adduct was formed: its formation requires the presence of cupric copper[7]. After treatment with phenylhydrazine the bovine plasma enzyme shows changes in the ESR parameters of only one copper [7], indicating that the two copper sites are not identical. The non-identity of copper sites was demonstrated also in the pig plasma enzyme by magnetic studies [5]. The copper atoms however, become equivalent after treatment with strong ligands like N 3− and CN− [5]. These results indicate a functional significance of the difference between the two copper atoms. Since the enzyme molecule contains two non identical copper ions and only carbonyl group, these findings suggest that only one of the two coppers has a role in the substrate oxidation. If the second prosthetic group is pyridoxal-5-phosphate, one of the copper ligands could be the nitrogen of the pyridine ring [1]. The substrate may bind the carbonyl cofactor and copper may have a catalytic role in oxygen activation [7].