Pig brain pyridoxal kinase and pyridoxime-5-phosphate oxidase

The inhibition kinetic patterns obtained when ATP and pyridoxal analogues are used as inhibitors of the reaction catalyzed by pyridoxal kinase are consistent with a rapid equilibrium random Bi-Bi, in which binary complexes, i.e., enzyme-ATP and enzyme-pyridoxal, are formed in kinetically significant amounts. Protein fluorescence quenching was used to determine the dissociation constant (K_D = 25µM) of ATP-Zn bound to the nucleotide site of the kinase. The binding of ATP to the kinase induces a conformational change which is transmitted to other areas of the macromolecule.

Pyridoxaloxime, a competitive inhibitor of pyridoxal, was used as a probe of the pyridoxal binding site. It binds to the kinase with a K_I = 2µM and displays a fluorescent decay time of 7.8 nanoseconds. Time emission anisotropy measurements yield a rotational correlation time for bound pyridoxaloxime of approximately 2 nanoseconds, which is considerably shorter than the rotational correlation time of the protein (Φ =38 nanoseconds). The fast rotation of pyridoxaloxime remains unaffected by the binding of ATP.

A rapid and simple method by which pig brain Pyridoxine-5-phosphate oxidase can be purified in high yield is described. m-Carboxyphenyl-pyridoxamine phosphate (CPP_p), an excellent substrate for pyridoxine-5-phosphate oxidase, was used as a fluorescent prode for the catalytic site. The fluorescence enhancement detected upon mixing the apoenzyme with Cpp_p suggested the interaction of this synthetic substrate and protein. This result indicated that the substrate recognized its binding site even in the absence of the cofactor FMN.