Pyridoxine-5-phosphate : identification of isozymes and sysnthesis of new substrates

Pyridoxine-5-phosphate oxidase was purified from pig liver. A coupled fluorometric enzymatic assay was developed. The assay offers increased sensitivity and the capability of continuously monitoring the time course of the reaction.

Polyacrylamide gel electrophoresis coupled with activity staining revealed the existence of more than one form of the oxidase. Further analysis revealed that these forms have similar molecular weights but vary in their charge to mass ratios.

Phosphopyridoxyl analogues were used to study properties of the active site. Aliphatic and aromatic carboxylic acids were attached to the primary amino group of pyridoxamine-5-phosphate. The aliphatic carboxylic acids ranged from three to seven methylene groups in length. The active site is capable of accommodating aliphatic carboxylic acids up to five methylene groups in length.

Phosphopyridoxyl-m-aminobenzoic acid yielded a two-fold increase in the k_cat and a ten-fold decrease in the K_M in comparison to the aliphatic carboxylic acid analogues. These dramatic changes in the kinetic parameters for phosphopyridoxyl-m-aminobenzoic acid are related to the effect of the aromatic ring on the amine. Alteration of the the electronic configuration may facilitate formation of the transition intermediate. Once formed, the intermediate is capable of being stabilized by the additional resonance energy offered by the aromatic group.

Separation distance between the bound FMN and the substrate binding site of the oxidase was determined by resonance energy transfer experiments. A value of 17 Å was obtained.