Study of the kinetic characteristics of d-amino acid oxidase using 2,2′-azinodi(3-ethylbenzothiazoline-6-sulphonic acid) as oxygen acceptor

Abstract

A simple kinetic method for assay of d-amino acid oxidase (d-AAO) catalytic activity using 2,2′-azinodi(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) as chromogen is described. Hydrogen peroxide produced in the reaction catalysed by d-AAO stoichiometrically oxidezes ABTS in a peroxidase-catalysed reaction. The absorbance of the chromogen measured at 410 nm is directly proportional to d-AAO activity. The estimated values of the Michaelis-Menten constants (Km) for d-alanine, d-proline, d-valine, d-2-aminobutyrate and d-leucine, determined in 100 mM pyrophosphate buffer (pH 8.8) at 30°C, were 2.60, 3.40, 4.60, 1.98 and 0.14 mM, respectively. The pH optima for the d-AAO activity was found to be between 8.6 and 8.8. The optimum temperature was about 30°C and the activation energy 8.89 kJ mol-1. The Km values for peroxidase, as an auxiliary enzyme in the indicator reaction, were 19.0, 20.8, 17.3 and 27.2 U 1-1 for d-alanine, d-proline, d-2-aminobutyrate and d-leucine, respectively, as substrate. The Km value for ABTS was 1.16 mM. The inhibition by sodium benzoate was found to be competitive with an inhibition constant, Ki, of 2.7 × 10-5 M. In all experiments, 50.5 μM of free flavine adenine dinucleptide was added to the reaction mixture to avoid dissociation of the holoenzyme.