The oxidation and reduction reactions of bovine serum amine oxidase. A kinetic study

Abstract

The presteady-state and steady-state kinetics of bovine serum amine oxidase (BSAO) were analyzed by stopped-flow transient spectroscopy. A simplified model of the catalytic cycle was found to describe the experimental data and the rate constants of the individual steps were used to calculate Michaelis parameters that agree with the direct determinations. In spite of many studies on selected reactions from the catalytic cycle, this is amongst the first efforts to provide a comprehensive kinetic description of the reactions of BSAO, whose results can be compared with the steady-state parameters. The reoxidation reaction by dioxygen is more complex than previously thought, in agreement with a recent report [Su, Q. and Klinman, J.P. (1998) Biochemistry 37, 12513-12525], and occurs in at least two steps whose rate constants, previously undetermined, have been measured. The reaction of the oxidized enzyme with the amine substrate is poorly determined in this type of experiment, thus irreversible combination with aromatic hydrazine inhibitors was used as a model system, demonstrating that the mechanism and rate constants of their reaction is fully compatible with an accurate description of the catalytic cycle with the physiological substrate. These results constitute a simplified, yet complete and consistent, description of the catalytic cycle and offer an interesting comparison with those obtained on plant amine oxidases; two steps of the catalytic cycle are significantly slower in BSAO than in pea seedling or lentil seedling amine oxidases, namely the reoxidation and the trans-iminative proton abstraction occurring in the enzyme-substrate complex. The former difference is rationalized as being due to the low to zero concentration of the semiquinolamine-radical intermediate, while the latter is less easily interpreted.