Mechanistic studies of reactions catalysed by diamine oxidase using isotope effects

Diamine oxidase (DAO), the enzyme that is responsible for amine biodegradation in animals, plants and humans, catalyses the biotransformation of amines such as histamine (HA), putrescine, 1-phenylethylamine, tyrosine, tryptamine, serotonine and spermine. The kinetic and solvent isotope effects (SIEs) were applied to study the mechanism of the biotransformation using HA and its methylderivatives. The SIE for the biotransformation of HA, N(τ)-methylhistamine and N(π)-methylhistamine was found to be 3.58, 2.22 and 5.70 on Vmax, and 1.58, 1.06 and 1.14 on Vmax/KM, respectively. On the other hand, the kinetic isotope effect for oxidation of stereospecifically deuterium-labelled [(α R)-(2)H]-N(τ)-methylhistamine and [(α R)-(2)H]-N(π)-methylhistamine was 0.69 and 0.62 on Vmax, and 15.06 and 7.50 on Vmax/K(M), respectively. These results demonstrate that DAO catalyses amine biotransformation by stereospecifically cleaving the αC-H bond in the pro-S position. Moreover, the oxidation of amine to aldehyde involves several transition states, including hybridisation change from sp(3) (Schiff base) to sp(2) (imine), then back again to sp(3) to give a final product with hybridisation sp(2) (aldehyde).