Lathyrus cicera copper amine oxidase reactions with tryptamine

Abstract

Lathyrus cicera copper amine oxidase (LCAO) rapidly formed the typical Cu(I)-TPQ semiquinone UV–visible spectrum, identical to that formed by other substrates, upon O2 exhaustion by turnover with excess tryptamine. A new band at 630nm formed more slowly, with intensity dependent on aldehyde and H2O2 concentrations. On prolonged incubation, all bands decayed in parallel, together with loss of enzymatic activity. The blue color disappeared on addition of KCN, a Cu(I) stabilizing agent, while the intensity of the radical visible bands increased. This shows that the 630nm absorbing species is a Cu(II) derivative, as confirmed by the unchanged intensity of the EPR spectrum of the frozen blue solution from that of the native protein. Rapid kinetics experiments showed that this species derives from a reduced form of the protein, plus aldehyde and H2O2 and that it is not in dynamic equilibrium with the radical. Given the similar population of the semiquinone radical with all substrates, it is possible that the reaction with aldehyde and H2O2 occurs in all cases although substrates lacking the indole group only produce the Cu(I)-semiquinone band. The radical participation to the catalytic activity is demonstrated by the observation that its relative population (controlled by the pH) parallels changes in the reoxidation rate constant, while the 630nm absorbing species is implied in the inactivation process, which depends on H2O2 and aldehyde concentration. The results of the paper are consistent with half-of-the-site reactivity, i.e. the two subunits of LCAO are kinetically and spectroscopically distinct from each other.