Studies on the enzymes pyridoxal kinase and pyridoxine phosphate oxidase

Pyridoxal kinase has been purified 200o-fold from pig brain. The enzyme preparation migrates as a single protein and activity band on analytical gel electrophoresis. The enzyme requires a divalent and a monovalent metal ion for its activity. Of the divalent metal ions studied, Zn ⁺² is the most effective in catalyzing the formation of pyridoxal-P from ATP and pyridoxal; the order of activation is Zn⁺² > Co ⁺²> Mn ⁺²> Mg ⁺² >Fe ⁺²> On the other hand, the monovalent cation required for enhancing its enzymatic activity is determined to be k†. Not only can pyridoxal kinase phosphorylate pyridoxal, it can also phosphorylate pyridoxine and pyridoxamine from ATP and Zn +2 ion. This enzyme is shown to contain one single subunit of molecular weight 60,000.

Different biogenic amines have been found in our laboratory to inhibit the pyridoxal kinase. By attaching a hydrophobic dansylated group (dansyl = 5-dimethylaminonaphthalene-l-sulfonyl) to some of the biogenic amines, the potency of inhibition is greatly enhanced. . The inhibitory level of γ-aminobutyric acid is increased by l000 fold after its modification to dansylated γ-aminobutyric acid. The biogenic amines show competitive inhhbition with respect to ATp and uncompetitive to pyridoxal.

The interactions of the substrate pyridoxal and inhibitor N-dansyl- 2-oxopyrrolidine with the catalytic site have been examined by means of fluorescence spectroscopy. The increase in emission anisotropy that follows the binding of pyridoxal to the kinase is used to determine the equilibrium constant. Pyridoxal kinase binds one molecule of substrate with K _d

N-dansyl-2-oxopyrrolidine is a competitive inhibitor of pyridoxal kinase with respect to ATp at saturating concentrations of pyridoxal. It binds to the enzyme with a dissociation constant of 6μM. N-dansyl-2-oxopyrrolidine is immobilized by strong interactions with the enzyme, but it is displaced from the catalytic site by ATP. The results are consistent with the hypothesis that N-dansyl-2oxopyrrolidine binds at the nucleotide binding site of pyridoxal kinase.

Irradiation of pyridoxal kinase in the presence of riboflavin leads to irreversible loss of catalytic activity. . Riboflavin binds to the kinase with K_d = 5uM without any inhibitory effect as shown by fluorometric titrations. Singlet excited oxygen, generated by energy transfer from the lowest triplet of riboflavin to oxygen, acts' as the oxidizing agent of approximately one histidine residue per mole of enzyme. The amino acid residues, such as tyrosine, tryptophan and cysteine are not photooxidized by the sensitizer bound to the enzyme. It is postulated that histidine is involved in the binding of the substrate ATp to the catalytic site of pyridoxal kinase.

Purification of pyridoxine-P oxidase has also been achieved from pig brain. The enzyme preparation is regarded as pure since it migrates as a single protein and activity band on the analytical gel electrophoresis. This enzyme catalyzes the oxidation of a primary

alcohol, a primary amine and a secondary amine. Molecular weight of the enzyme is determined to be 62,000 by sucrose density gradient centrifugation and sodium dodecyl sulfate polyacrylamide gel electrophoresis. In addition, sodium dodecyl sulfate polyacrylamide gel electrophoresis reveals that pyridoxine-P oxidase consists of two identical subunits of molecular weight 31,000 each. Pyridoxine-P oxidase, like many other oxidases, requires FMN as the cofactor for its enzymatic activities. Pyridoxine-P oxidase can be resolved from the holoenzyme to its apo- form and active holoenzyme can be reformed by addition of exogenous FMN to the apoenzyme. One sulfhydryl group per molecule of enzyme is titrated by DTNB on pyridoxine-P oxidase without any loss of enzymatic activity. Attachment of fluorescent probe, such as jodoacetamide fluorescein, on pyridoxine-P oxidase does not affect its catalytic activity.

Complex formation between pyridoxal kinase and pyridoxine-P oxidase is detected; the results of the energy transfer experiment reveal that the two enzymes are approximately 35A apart in solution. Increase of polarization of fluorescence from 0.2 to 0.25 further confirm the formation of the protein-protein complex when pyridoxine-P oxidase is mixed with equal molar concentration of pyridoxal kinase. Further evidence supporting protein-protein interaction was obtained by Sephadex G-100 chromatography, sucrose density gradient centrifugation, and transient kinetic' analysis of the coupled assay system. The kinetic analysis used pyridoxine as the substrate for pyridoxal kinase and then pyridoxine-P oxidase continued the oxidation reaction to form pyridoxal-P.

Reduction of the transient time for the coupled assay system provides evidence for the possibility of interaction between the two enzymes. A functional role played by the enzyme-enzyme complex in the regulation of accumulation of pyridoxal-p is discussed.