Brain pyridoxine-5-phosphate oxidase. A dimeric enzyme containing one FMN site.

Pyridoxine-5-phosphate oxidase has been purified 2250-fold from pig brain by affinity chromatography. The enzyme of 60000 molecular weight is made up of two identical size subunits and binds 1 mol of FMN/mol dimer. 1 mol of the fluorescent inhibitor phospho-pyridoxal oxime interacts with 1 mol of the dimeric protein to yield a dissociation constant KD = 0.2 microM. Resolution of the holoenzyme into apoprotein and free FMN does not induce dissociation of the dimeric structure. The oxidase catalyzes the oxidation of the natural substrates pyridoxine 5-phosphate and pyridoxamine 5-phosphate, but phospho-pyridoxyl derivatives of the aromatic carboxylic acids, p-aminobenzoate and m-aminobenzoate, are also excellent substrates of the enzyme. Introduction of electron-withdrawing substituents into the structure of benzene increases the kcat values. A comparison of the kcat values obtained with several synthetic substrates suggests that electron-withdrawing substituents tend to stabilize carbanionic intermediates formed in the earlier stages of catalysis.