Spectral and kinetic studies on Pseudomonas L-phenylalanine oxidase (deaminating and decarboxylating)

Pseudomonas L-phenylalanine oxidase (deaminating and decarboxylating) contains two FAD molecules in one molecule of the enzyme (Koyama, H. (1983) J. Biochem. 93, 1313-1319). When the enzyme was mixed anaerobically with L-phenylalanine, beta-2-thienylalanine, L-tyrosine, or L-methionine, a spectral species (purple intermediate) with a broad absorption band around 540 nm was observed with each substrate, and decayed slowly. From the data on the overall reaction kinetics, the rate of the L-phenylalanine oxidase reaction was expressed as follows. e/v = e/Vm + A/[S] + B/[O2] where e represents the concentration of enzyme unit, v the rate of the overall reaction, Vm the maximum velocity, and A and B are constants. Furthermore, the reactions of the enzyme with beta-2-thienylalanine (mostly an oxygenase substrate) and L-methionine (an oxidase substrate) were analyzed by the "stopped flow" method. The following scheme for the mechanism of L-phenylalanine oxidase reaction with both substrates is proposed, based on the data obtained. (formula; see text) Where Eox represents the oxidized form of the enzyme unit, EoxS the enzyme unit (oxidized form)-substrate compound, X the purple intermediate with a characteristic broad absorption band around 540 nm, S the substrate and P the product.