Kinetic Studies on the Inhibition Mechanism of Diamine Oxidase from Porcine Kidney by Aminoguanidine

The mechanism of inhibition of diamine oxidase [DAO, histaminase, amine: oxygen oxidoreductase (deaminating) (copper-containing), EC 1.4.3.6] by aminoguanidine was studied kinetically. The DAO reaction was carried out with putrescine as a substrate in the presence of o-aminobenzaldehyde and followed by the continuous increase in A430. An enzymatic reaction product, 4-aminobutyraldehyde, was spontaneously cyclized to Δ1-pyrroline, which coupled with o-aminobenzaldehyde to give rise to a yellow quin-azolinium chromophore [Holmstedt et al. (1961) Biochim. Biophys. Acta 48, 182–186]. First, to measure the initial velocities as accurately as possible, the kinetic relationship between the oxidase reaction rate and the color development rate was formulated. The reaction proceeded linearly in the absence of aminoguanidine, but slowed down time-dependently in its presence. When the color development velocities in the presence of the inhibitor were plotted against time, the plot was nonlinear and there seemed to be at least two phases of the inhibition. Nonlinear least-squares analysis of the data dA450/dt showed that the simplest model that fitted best was a model composed of two species of enzyme-inhibitor complexes: where E is the enzyme, I is aminoguanidine (an inhibitor), and the As are the rate constants. The kinetic parameters (at pH 7.4, 30°C) were determined to be as follows: k+1, =(2.5±0.3) × 104 M−1,8−1, k−1=(1.3±0.5)×10−3 8−1, k+2 =(2.2±0.5)×10−3 8−1 k−2 (3.0±0.7)×10−5 8−1. Because the k−2- value is only one-seventieth of the k+2 value and thus the E'I species is the most favorable on equilibrium, aminoguanidine not only inhibits DAO but also apparently inactivates the enzyme.