title: Brain pyridoxal kinase. Mechanism of substrate addition, binding of ATP, and rotational mobility of the inhibitor pyridoxaloxime.
abstract: 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 (Kd = 25 microM) 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 Ki = 2 microM and displays a fluorescent decay time of 7.8 ns. Time emission anisotropy measurements yield a rotational correlation time for bound pyridoxaloxime of approximately 2 ns, which is considerably shorter than the rotational correlation time of the protein (phi = 38 ns). The fast rotation of pyridoxaloxime remains unaffected by the binding of ATP.
mesh_terms: D000255:Adenosine Triphosphate; D000818:Animals; D001921:Brain / Q000201:enzymology*; D007700:Kinetics; D010770:Phosphotransferases / Q000378:metabolism*; D011485:Protein Binding; D011730:Pyridoxal / Q000031:analogs & derivatives* / Q000494:pharmacology; D011731:Pyridoxal Kinase / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013552:Swine
chemical_list: D011730:Pyridoxal; C028073:pyridoxaloxime; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D011731:Pyridoxal Kinase