Brain pyridoxine‐5‐phosphate oxidase

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Abstract
Pyridoxine-5-phosphate oxidase was purified 2250-fold from pig brain by affinity chromatography. The enzyme of 60,000 MW is made up of 2 identical size subunits and binds 1 mol of FMN/mol [flavine mononucleotide] dimer. One molecule of fluorescent inhibitor phospho-pyridoxal oxime interacts with 1 mol of the dimeric protein to yield a dissociation constant Kd = 0.2 .mu.M. 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.