Purification and Properties of Pyridoxamine (Pyridoxine) 5'-Phosphate Oxidase from Baker's Yeast

Abstract

Pyridoxamine (pyridoxine) 5'-phosphate oxidase (EC. 1.4.3.5) has been purified from dry baker's yeast to an apparent homogeneity on a polyacrylamide disc gel electrophoresis in the presence of 10 μM of phenylmethylsulfonyl fluoride throughout purification.
1) The purified enzyme, obtained as holo-flavoprotein, has a specific activity of 27μmol/ mg/hr for pyridoxamine 5'-phosphate at 37°C, and a ratio of pyridoxine 5'-phosphate oxidase to pyridoxamine 5'-phosphate oxidase is approximately 0.25 at a substrate concentration of 285μm. Km values for both substrates are 18μM for pyridoxamine 5'-phosphate and 2.7μm for pyridoxine 5'-phosphate, respectively.
2) The enzyme can easily oxidize pyridoxamine 5'-phosphate, but when pyridoxamine and pyridoxine 5'-phosphate are coexisted in a reaction mixture the enzyme activity is markedly suppressed much beyond the values expected from its high affinity (low Km) and low V_max for the latter substrate.
3) Optimum temperature for both substrates is approximately 45°C, and optimum pH is near 9 for pyridoxamine 5'-phosphate and 8 for pyridoxine 5'-phosphate.
4) From the data obtained, the mechanism of regulation of this enzyme in production of pyridoxal 5'-phosphate and a reasonable substrate for the enzyme in vivo are discussed.