Brain pyridoxal kinase. Mechanism of substrate addition, binding of ATP, and rotational mobility of the inhibitor pyridoxaloxime.
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.
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
D011730:Pyridoxal; C028073:pyridoxaloxime; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D011731:Pyridoxal Kinase