Brain pyridoxal kinase. Mobility of the substrate pyridoxal and binding of inhibitors to the nucleotide site.
Pyridoxal kinase has been purified 2000-fold from pig brain. The enzyme preparation migrates as a single protein and activity band on analytical gel electrophoresis. The interactions of the substrate pyridoxal and the inhibitor N-dansyl-2-oxopyrrolidine (dansyl = 5-dimethylaminonaphthalene-1-sulfonyl) with the catalytic site were examined by means of fluorescence spectroscopy. The increase in emission anisotropy that follows the binding of pyridoxal to the kinase was used to determine the equilibrium dissociation constant. Pyridoxal kinase binds one molecule of substrate with a Kd = 11 microns at pH 6. The emission anisotropy spectrum of bound pyridoxal reveals that the substrate is not rigidly trapped by the protein matrix. N-Dansyl-2-oxopyrrolidine is a competitive inhibitor with respect to ATP at saturating concentrations of pyridoxal. It binds to the enzyme with a dissociation constant of 6 microns. 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-2-oxopyrrolidine binds at the nucleotide binding site of pyridoxal kinase.
D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D003619:Dansyl Compounds / Q000494:pharmacology; D007700:Kinetics; D010770:Phosphotransferases / Q000378:metabolism*; D011485:Protein Binding; D011730:Pyridoxal; D011731:Pyridoxal Kinase / Q000302:isolation & purification / Q000378:metabolism*; D011760:Pyrrolidinones / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013552:Swine
D003619:Dansyl Compounds; D011760:Pyrrolidinones; D011730:Pyridoxal; C018888:N-dansyl-2-oxopyrrolidine; D010770:Phosphotransferases; D011731:Pyridoxal Kinase